CA2143336A1 - Tissue specific transcriptional regulatory element - Google Patents

Abstract

The present invention therefore provides a transcriptional regulatory element which is capable of directing expression of a gene specifically in cells of the endothelial lineage. Preferably, the transcriptional regulatory element comprises the initiation cod-on and the untranslated sequence of tek, a protein tyrosine kinase expressed during murine cardiogenesis and homologues there-of including the human tek gene. The transcriptional regulatory element may be used to target expression of a gene in cells of the endothelial lineage.

Description

~ YLle Uo~ b~
~ 2143336 Title: TISS~E ~P}~CIFI~
~RA~SCRI~rI~NAI. R}~Ç;~OR~ ~r.

FI~IJr) OF T~B lhvlsN ~ ION
The invention re~ ata~ to a novel tran~cripticnal 5 regul~tory element ~h~ ch i3 capable c~ directing expresslon o~ a gene spec~ ~icaLly ~n cells o~ the e~dotheli~l lineage; a ~ecombinant molecule cent~ining ~he ~ranscription~l regula~ory element; a tran~fo~nant h~st cell including ~he -eco~nbinar.t moleculef a D~A construct 10 co~p;:l~ing th~ transcriptional regulatory element operat~eLy lin}ced to a gene and~ ~ reE?orter gene; a~d, ~he use of the tran~criptional regulat~ry element ~o ~arget ex~res~on of ~ gene in celLs of the endothel~al lineage.

P~At~ Our~ OF ~R l~v~ ON
~is~ue ~pecif ic transcriptiana~ regul~tary element have ~een identif ied w}~ch have been used to target expres~ion of exogenous gene~ in cells ~nd in transge~ic ~n1m~1 g, F~r example, ~S~A constructs usirlg an erythrc>i~ ~peclfic transcrip~LorL~l elem~nt and ~n oncogene 20 encoding ~ pr~tein having prot~in-tyroslne kinase (P~K~
activity ~ave been used to prZ~duce tran~genic animals w~lch have carc~io~scular disease ~Yee, S.P. e~ al. ~198g) P.~.A.S., ~.S.A. 86, ;8~3-877).
The a~ility to introduce into A n i rt;~ 1 S exogerlous 25 genes whi ch ~re se~ erti~rely expre~sed in a particul~r cell type pro~7ides wide ranging experimental as well as practiczl op~rtun~ ties . II1 p~rt cul2r it permit~
inv~stlgat~ion o the rol~ cDf a subs~ance in ~he de-Jelopmen~, dete~minati~n, migration, or proliferation o~
30 cells c:lf a partic~llar lineag~.
Dumont e~ al (OIlcogene ~l~S~), 7:1~71-1480) repor~ the i~olation of a novel tyro~ine kinase~
dec ignated tek which maps to mouse chro~r.osome 4 between ~he brown alld pm~r- 2 3 1 oc i . They ~ howeci by ~ n s i tu 3 ~ hybridizat ~ on tha~ tek i8 expressed ~ n ~he endocardillm as AMENDED S~EET

~ 2143336 - lA -weLl as the en~thel ~ al linir g o~ the ~asc~La~ure .

S~MARY C~F TH~ lNV ~ ~'l'iO~
~ he pres~nt ~ n~entors h~ve ider~ ~ied transcripticnal regul~tor~ eiement character~ zed ~y endothelial fipecific expre~sion- The element i5 expressed ~n cells of ~he endo~heli~l lineage inc~ udin~ mature and pro~en~tor cells. ~his i~ the first repo ~ c AMEND D SH~EI

W094/~K94 PCT/CA93/00352 -

2~3336 _ - 2 -transcriptional regulatory element which is capable of directing expression specifically in cells of the endothel i Al lineage.
The present invention therefore pro~ides a transcript~ O~A 1 regulatory element which i~ capable of directing expression of a gene specifically in cells of the endo~h~l;Al 1 in~Age- Preferably, the transcriptionsl regulatory element comprises the initiation codon and the untranslated sequences of tek, a protein tyrosine kinase expressed during murine cardiogene~ ~nd homologues thereof including the human tek gene. Most preferably, the transcriptional regul~tory element i~ a 7.2 kb fragment exten~ i ng from the Bgl II site to the ~pn I site as shown in Figur~ 3.
15The invention further provide~ ~ method of = preparing the transcriptional regulatory element. The transcripti 0~1 regul~tory element ~y be constructed by synthesis and ligation of DNA oligomer~. The element may also be i~olated by selectively amplifying the region of the transcript~o~Al regulatory elemert using the polymerase chain reaction method and genomic DNA.
The invention also permit~ the construction of nucleotide probes which are unique to the transcrip~;onAl regulatory element of the invention. Thus, the invention al~o relateQ to a probe comprising a nucleotide ~equence substantially homologous to the transcriptional regulatory =element of the invention. The probe may be lAhelled, for example, with a rA~io~ctive suhstance and it may be used to select from a mixture of nucleotide se~e~re~ a transcription~l regulatory element of the in~ention or an element homologous thereto.
The invention also relates to a recomhinAnt molecule adapted for transformation of a ho~t cell comprisinq a transcriptionAl requlatory element of the invention and a gene operatively 1~nke~ to the transcrip~io~l regulatory element. A transformant host cell including a recombinant molecule of the irvention is ~ 214 3 3 3 6 PCT/CA93/00352 also provided. Still further, this invention provides plasmids which comprise the transcriptional regulatory element of the invention.
In an embodiment of the invention a recombinant molecule comprising the transcriptio~Al regulatory element of the invention operatively 1 in~e~ to a gene and a e~olLer gene is provided.
The recombinant molecules of the invention may be used ~o produce transgenic non-human mammals.
Accordingly the invention relates to a method of producing a transgenic non-human mammal characterized as having a plurslity of cells cont~i n i ng a recombinant molecule of the in~ention, or an ancestor of the mammal at an embryonic stsge, comprising (a) introducing the recombinant molecule into a pronucleus of a ~ammalian zygote by microin~ection, ~aid zygote being capable of development into ~ mammal, thereby obtAini~ a genetic~lly transformed zygote; (b) transplanting an embryo derived from the geneti~Ally transformed zygote into a pseudo-pregnant female o~r~hle of bearing the embryo to term and(c) if deæired allowing the embryo to develop to term.
The invention further relates to a transgenic non-human mammal all of whose germ cells and somatic cells contain a recom~jn~nt molecule of the invention introduced into the mammal, or an ancestor of the mammal at an embryonic stage. Still further the invention relates to cell cultures of cells of the transgenic mammals.
The invention also relates to a method of determining the affect of a substance on cells of the endothp~ in~ge comprising producing a transgenic non--~ human mammal characterized a8 having a plurality of cells cont~i n i ng a recombinant molecule comprising the transcripti n~ 1 regulatory element of the invention operatively linke~ to a gene encoding the ~ubstance, or an ance tor of the mammal at an embryonic st ge, comprising (a) il-Lloducing the recombinant molecule into a pronucleus of a mammalian zygote by microin~ection, said zygote being PCI`/CA93/0035 capable of development into a mammal, thereby obt~inin~ a genetically transformed zygote; (b) transplanting an embryo derived from the genetically transformed zygote into a pseudo-pregnant female capable of bearing the embryo to term and (c) isolating the.embryo or allowing the embryo to develop to term, and (d) determining the affect of the substance on cells of the endotheli~l lineage by comparison to a control.
In an embodiment of the invention, a method of determining the affect of a substance on cells of the endothelial lineage is provided comprising producing a transgenic non-human mammal characterized as having a plurality of cells cont~in;ng a recombinant molecule comprising the transcriptionAl regulatory element of the invention operatively linke~ to a gene ~nd a ~e~o,Ler gene ~nro~ing a phenotype which is not displayed by the mammal, or an ancestor of the mammal at an embryonic stage, comprising (a) introducing the recombinant molecule into a pronucleus of a mammalian zygote by microin~ection, said zygote being capable of development into a mammal, therQby obt~in~ng a gene~irAlly trsnsformed zygote; (b) transpl2nting an embryo derived from the genetically transformed zygote into a pseudo-pregnant female capable of bearing the embryo to term and (c) isolating the embryo or allowing the embryo to develop to term, (d) assaying for the phenotype of the .e~o~Ler gene in the embryo or transgenic non-human mammal to determine the pattern and extent of expression of the gene, ~nd (e) determining the affect of the substance on cells of the endothelial linPAge by comparison to a conL.ol.
n~RTPTION 0~ ~R~ DRA~TN~-~
The invention will be better understood withreference to the drawings in which:
Figure 1 shows the nucleotide and deduced a~ino acid sequence of tek;
Figure 2 shows the nucleotide and ~P~t~ced amino sequence of a 1601 bp DNA segment of tek Figure 3 is a restriction map showing the transcriptio~Al regulatory element of the invention fused to reporter gene LacZ;
Figure 4 is a schematic diagram showing the predicted structure of tek;
Figure 5 shows a Northern blot hybridization analysis of expression of tek in 12.5 day murine emLlyo~ic heart;
Figure 6 shows the in situ hybridization analysis of ex~ession of tek in the 12.5 day embryo;
Pigure 7 shows the expression of tek precedes that of von Willebrand factor in 8.5 day emhryos;
Figure 8 shows expression of tek in whole mount embryos(A., B., and C.); ex~e~ion in Day 8.0 embryos (D.); mRNA di~tribution in a Day 9.5 embryo (E.); snd En2 expression in a Day 8 embryo (F.);
Figure 9 shows the ~ Bsion of tek ~l~C~
that of von W~ hrand factor in the developing leptomen ~ n~ e ~ and in particular the absence of immunohi~tochemical st~ininq of von Willebrand factor in Day 12.5 leptomeninges (A); in situ detection of tek expression in Day 12.5 leptomening~(B); stAining of von Willebrand factor in Day 14.5 leptomeninges tC);
Figure 10 shows the ~ e~sion of tek in adult vasculature and in particular bright field illumination of a section through the upper heart region of a 3 week-old mouse hybridized with an t35S]-l~h~l led tek probe (B);
bright field illumination showing tek expression in endoth~ l cell~ l~ning the artery and vein respectively 30 (C);
- Figure 11 ~hows the partial nucleotide sequence of the tran~criptin~A1 regulatory element of the - invention;
Figure 12 show~ expression of LacZ in Day 8.5 embryos pro~l~ce~ using a DNA construct comprising the transcriptionA1 regulatory element of the invention and LacZ; and Figure 13 shows tek mRNA distribution in a Day 8.5 embryo;
Figure 14 shows a compari~on of a portion of the deduced amino acid sequence of the novellreceptor tyrosine kinase protein of the invention with that of other tyrosine kinases. ~
n~ATT.~n nR~rRTpTIoN OF T~R TNV~NT~N
CopenAing application Serial No. 07/921,795 relates to a novel protein tyrosine kinase expre~sed during murine cardiogenesis which i8 designated tek. The human homolog of tek has been recently reported by Ziegler, G. F. et al., Oncogene 8s663-670, (1993). The te~ locu~ was mapped to chromo~ome 4, beL.Jeen the brown and pmv-23 loci. This region is ~yntenic with human chromosomal re~i Qn~ lp22-32, 9q31-33, and 9p22-13. The deduced amino acid sequence of tek predicts that it a putative e~-~Lor tyro3ine kinase that contains a 21 amino acid ki n~e insert and which is most closely related in its catalytic domain to FGFR1 (mouse fibroblast growth factor) and the product of the ret proto-oncogene.
Figure 1 shows the nucleotide and ~e~nce~ amino acid seguence of tek. The present inventors have also identifi~ the initiation site of translation of tek.
In the Adult ~nd ~ll stages of embryonic development examined, tek expression was found to be restricted to cells of the endo~h~ n~ge.
Specifically, in situ hybridization analysis of adult tissues, a~ well as ~ectio~ and whole mount embryos, showed that tek is 3pecifically expre~sed in the en~or~rdium, the leptomeninges and the endoth~liAl lin~ng of the vasculature from the earliest stages of their development. Mo e~ve , examination of the morphology of tek-ex~ essing cells, and 8tAgi ng of tek expres~ion relative to that of the endoth~l ;A1 cell marker von ~illebrand factor, revealed that tek is expressed prior to von Will~h~and factor and ~ppears to mark the embryonic ~oye~itor~ of mature endo~h~ l cell~.

214 3 3 3 6 PCT/CA93/~3~2 The present inventors have identified a transcriptional regulatory element located upstream of tek which specifically directs expression of a gene in cells of the endotheli A 1 l i n~Age The transcriptionAl regulatory element has been found to direct expresfiion in both mature and ~Gye--itor endothelial cell~.
In particular, the present inventors isolated a DNA segment from a mouse genomic bacteriophage library using a 5'-prime probe which contA i n~A the initiation codon and untranslated sequences of tek using the procedures of Sambrook et al., 1989, Nolecular Cloning, A
Laboratory MAn~l. Cold Spring Harbour Lab. Press. A 16 kb phage clone was shown by hybridization and sequence analysis to contain a single exon with 175 bp homologous to the cDNA. ~ DNA fragment ext~ ng from the Bgl II
restriction site located at nucleotide 110 of the cDNA to the nearest Kpn I in the phage was cloned Up8 tre~m of the bacterial gene lacZ (see Figure 3). This reporter construct cont~i n i ng 7.2 kb of the tek gene wa8 microin~ected into pronuclei of fertil;~ randomly ~red CD-l mice using plo~edures as set out in ~og~n et al.
(1986, Manipulating the Mouse Embryo, A Laboratory MAnn~l.
Cold Spring ~Arhor Lab. Press). Three transgenic founder embryos were dis~ected from their foster mothers and yolk sac DNA analyzed for the presence of the transgene.
Expre~sion of the transgene was determined by the X-gal st~ining of whole embryos and subsequent sectio~ing of the embryos. The 7.2 kb fragment was able to drive lacZ
e~ sion in endo~hel~l cells that had previously been shown to express tek RNA thus demonstrating that this DNA
contAin~ the tek transcription~l regulatory element. The partial nucleotide sequence of the transcriptional regulatory element i8 shown in Figure 11.
The invention further provides a method of preparing the transcrip~ l regulatory element. The tran8cription~ 1 regulatory element may be isolated by selectively amplifying the region of the transcriptional _ ' ` PCT/CA93/00352 regulatory element using the polymerase chain reaction method and genomic DNA. It is possible to design synthetic oligonucleotide primers from the sequence shown in Figure 11 for use in PCR and for scr~e~i n~ genomic libraries, in particular human genomic libraries. An amplified fragment can be cloned and characterized by DNA sequence analysis.
The nucleotide sequence of t~e transcriptional regulatory element will also permit the element to be constructed by synthesis and ligation of DNA oligomers. The transcriptional regulatory element may be ~Loven functional by sssessing the transient expression of a construct bearing a reporter gene. For example, using the reporter gene for B-galactosidase (LacZ) or chloramphenicol acetyltransferase (CAT) after transfection of the DNA into host cells.
It will be ap~Leciated that the invention includes nucleotide se~e~rP~ which have æubstantial sequence homology with the nucleotide sequence of the transcript; O~A 1 regulatory element of the invention. The term "se~Pnr-P~ h~ving sub~tantial sequence homology"
means those se~Pnces which have slight or inron~equential sequence variations i.e. the homologous se~e~ces function in substantially the same manner to produce substantially the same result as the actual sequence. The variations may be attributable to local mutations or structural moA;f;rAtions.
The invention also permits the construction of nucleotide probes which are unique to the transcriptional regulatory element of the invention. Thus, the invention also relates to a pro~e comprising a nucleotide sequence substantially homologous to the transcript;o~l regulatory element of the invention. The probe may be lAhelled and it may be used to select from a mixture of nucleotide se~en~ 8 tran8cript~A 1 regulatory element of the invention or an element substantially homologous thereto.
A nucleotide probe may be 1 ~hPl 1 ed with 8 rA~;oActive label which provides for an adequate signal and has 2Iq3336 094/~94 PCT/CA93/~352 sufficient half-life such as 32p~ 3H, ~4C or the like.
Other labels which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enz~mes, antiho~ies specific for ~ labelled antigen, and chemiluminescent substances. An appropriate label may be ~elected having regard to the rate of hybridization and bin~ing of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization.
The invention also relate~ to a recombinsnt molecule adapted for transformation of a host cell comprising a transcriptional regul~tory element of the invention and a gene operatively l~n~e~ thereto. The transcripti O~A 1 regulatory element of the invention operatively lirkeA to a gene may bQ ~o.~o ated in a known manner into a recombinant mol~cule which en~ures good expres~ion of the protein encod~d by the gene. The transcriptiQn~l regulatory element of the invention may be inco.~u.aLed into a plasmid vector, for example, a retroviral vector, pECE.
The transcriptiQnAl regulatory element of the invention may be operatively linke~ to a reporter gene or a gene enro~in~ a substance which has toxic or therapeutic activity including a factor which modulates angiogenesi~.
Examples of e~o Ler genes, factors which modulate angiogenesi~, and substances with toxic or therapeutic activity are di~cussed below.
A transformant host cell including a recombinant molecule o~ the invention and 8 cell line cont~i n i ng such transformant host cells i~ also provided. Examples of suitable ho3t cells include human endothelial cells such as umbilirAl v~in endoth~l 1A1 cells and rabbit aortic endothel i~l cells.
The invention al80 relatQ8 to a recombinant molecule comprising a transcripti Q~ 1 regulatory element of the invention operatively li nk~ to a gene and a e~o Ler gene. The reporter gene may be introduced into WO 94/~K94 PCT/CA93/003~2 ~

~43~ o-the recombinant molecule using conventional methods such as those described in Sambrook et al., 1989, Molecular Cloning, A Laboratory MAn~l. Cold Spring Harbour Lab.
Press. The recombinant molecule may also be ~ynthetically produced using co~,ve,.Lional methods. Further, the recombinant molecule may be introduc2d into a host cell using co~lven~ion~l methods. ~~
The reporter gene should be under the control of the transcription~l regulatory element and the pattern and extent of expression of the gene operatively linked to the tran3cripti O~A 1 regulatory element may accordingly be determined in cells of the endo~heliAl lineage. Preferably the epolLer gene codes for a phenotype not displayed by the host cell and the phenotype m~y be assayed quantitatively. Examples of suitable L~orLer genes include lacZ (B-galactosidase), neo (neomycin phosphotransfera~e), c~t (chloramphenicol acetyltransfers~e) dhfr (dihydrofolate reductase), aphIV
(hygromycin phosphotransfera~e), lux (luciferase), uidA
(B-glucuro~i~Ar?). Preferably, the reporter gene is lacZ
which codes for B-galactosidase. B-galactosidase may be assayed using the lactose analogue X-gal(5-bromo-4-chloro-

3-indolyl-b-D-galactopyranoside) which is broken down by B-galactosidas~ to a ~ oducL that is blue in color. (See for example Old R.W. & PrLmrose S.B., Principles of Gene MAniplllAtion An Introduction to Genetic Engineering, 4th ed. Oxford University Press at pages 63-66 for a discus~ion of procedures for screening for recombinant~).
~ he recombinant DNA of the invention may be used to produce transgenic non-human mamm~l~. Accordingly the invention relates to a method of producing a transgenic non-human mammal characterized as having a plurality of cells cont~inin~ a recombinant molecule of the invention, or an ancestor of the mammal at an embryonic stage, comprising (a) introducing the recombinant molecule into a pronucleu~ of a mam~AliAn zygote by microin~ection, said zygote being capable of development into a mammal, thereby ~ o 21~3336 94/~94 PCT/CA93/00352 obt~ining a genetically transformed zygote; (b) transplanting an embryo derived from the ~genetically transformed zygote into a pseudo- pregnant female capable of bearing the embryo to term and (c) if desired, allowing the embryo to develop to term.
The invention further relstes to a transgenic non-human mammal all of whose germ cells and ~omatic cells contsin a recombinant molecule of the invention introduced into the animal, or an ancestor of the mammal at an embryonic stage.
In a preferred embodiment, plasmids cont~i n i ng recombinant molecules of the invention (for example see Figure 3) are microin~ected into mouse embryos. In particular, the plasmid~ ~re in~ected into the male pronuclei of fertili7ed one-cell mouse eggs; the in~ected eggs are transferred to pseudo-pregnant foster females;
and, the eggs in the foster females are allowed to develop to term. (Hogan, B. et al, (1986) A Laboratory ~m~
Cold Spring uArhOr, New York, Cold Spring ~Arhor Laboratory).
It ~ill be re~ e~ that methods other than microin~ection can be u~ed to generate the transgenic mammals. For instance, retrovirus infection techniques (R. JA~ni~ch, PNAS U.S.A. 73, p. 1260 (1976); Cell 12, P.
691 (1977); H. Varmus, in RNA Tumor Viruses, R. Weis~ et al, Cold Spring U~rhor L horatory, Cold Spring U~rhor~ NY~
(1982) p. 369-512; D. Jahner and R. Scienisch, Nature 287, p. 456 (1980) and R. Jaenisch et al, Cell 24, p. 519 (1981)), direct introduction of DNA into sperm cells followed by in vitro fertili7~tion (Lavitrano, N., et al, - Cell. 57, p. 717), and techniques involving the introduction of DNA by viral transduction or transfection r into embryonic stem cells which are able to contribute to the germ line when in~ected into host blastocysts can be employed (A. Bradley et al, Nature 309, p. 255 (1984); A.
Gossler et al, PNAS U.S.A. 83, p. 9065 (1986)).
Although experimental animals used in the W094/~94 PCT/CA93/00352 2i 4333~

preferred embodiment disclosed were mice, the invention should not be limited thereto. It may be desirable to use other species such as rats, hamsters and rabbits.
The invention also relates to a method of determining the affect of a substa~nce on cells of the endothe~ ineAge comprising pro ~cing a transgenic non-human mammal characterized as haY~g a plurality of cells contAin;ng a recombinant molecule comprising the transcrip~io~l regulatory element of the invention operatively link~A to a gene, or an ancestor of the mammal at an embryonic stage, comprising (a) introducing the recombinant molecule into a pronucleus of a mammalian zygote by microin~ection, said zygote being capable of development into a mammal, thereby obt~ining a genetically transformed zygote; (b) transplanting an embryo-derived from the geneticAlly transformed zygote into a pseudo-pregnant female cApAhl~ of bearing the embryo to term and (c) isolating the embryo or allowing the embryo to develop to term, and (d) determining the affect of the ~ubstance on cells of the endoth~ in~Age by comparison to a control.
In an embodiment of the invention a method of determining the affect of a substance on cells of the endo~h~l~Al lineAge i~ provided comprising producing a transgenic non-human mammal characterized as having a plurality of cells cont~ini ng a recombinant molecule comprising a transcriptionAl regulatory element of the invention linkeA to a gene encoding the substance, and h ~e~u~er gene enroA;ng a phenotype which i8 not displayed by the mammal, or an ancestor of the mammal at an embryonic stage, comprising (a) introducing the recombinant molecule into a pronuclQus of a mammalian zygote by microin~ection, 8aid zygote being capable of development into a mammal, thereby obtAin;ng a genet~c~l1y tran~formed zygote; (b) transplanti ng an embryo derived from the genet;c~lly transformed zygote into a p~eudo pregnant female capable of bearing the embryo to term and 2143~36 ~ 094/~94 PCr/CA93/00352 :

(c) if desired, allowing the embryo to develop to term, (d) afisaying for the phenotype of the reporter gene in the embryo or transgenic mammal to determine the pattern and extent of expression of the gene, and (e) determining the affect of the substance on cells of the endothelial in~e by comparison to a st~n~rd.
As discussed above, the reporter gene should be under the control of the transcriptional regulatory element and accordingly the pattern snd extent of expression o a gene oper_tively linke~ to the transcriptional regulatory element may be determined by assaying for the phenotype of the reporter gene.
Preferably the e~olLer gene codes for a phenotype not displayed by the host cell and the phenotype may be assayed quantitatively. Examples of suitable reporter genes include lacZ (~-galactosidase), neo (neomycin phophotransferase), cat (chlor_mphenicol acetyltransferase) dhfr (dihydrofolate reductase), aphIV
(hygromycin phosphotransferase), lux (luciferase), uidA
(~-glucuroniAA~e). Prefersbly, the e~o Ler gene is lacZ
which codes for ~-galactosidase. ~-galactosidase may be assayed using the lactose analogue X-gal(5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside) which i~ broken down by ~-galactosidase to a product that is blue in color. (See for example Old R.W. & Primrose S.B., Principles of Gene Manipulation A~ Introduction to Genetic Engineering, 4th ed. Oxford University Press at pages 63-66 for a discussion of pro~e~res for scr~ening for recombinants).
Cells of the transgenic mammals of the invention and pro~ e~ by the methods of the invention may be cultured using stAnAArd tissue culture techniques.
The present invention allows the manipulation of - endo~h~ l cell physiology by targeting expression of a ~ubst_nce in cells of the endoth~ l lineage in a mammal.
The above described methods, transgenic animAls snd cell cultures derived therefrom, can therefore be used to assess the role of _ ~ubstance in the determination, W094/~K94 336 PCT/CA93/00352 -migration, or proliferation of cells of the endoth~l iAl lineage. In particular, the invention provides a me~hnni~m for investigating vascularization of tumors and the control of angiogenesis. A transgenic m_mmal may be produced which expres~es a substance exclusively in cell~
of the endo~h~l; A 1 1 i n~Age . A comparison of endo~h~
phenotype, morphology, and function using for example imm~lnohistochemical techniques and ass~ys for ~DL
receptors, and of the pattern and extent of expression of the substance in the animal with a control transgenic animal will provide an indication of the affect of the substance on cells of the endo~h~ l lineage.
Substances which may modulate the angiogenic process (herein also referred to ac angiogenic factors) may be tested using the above described method. Examples of such subst~nc~ include substances derived from human and animal tissues which stimulate the proliferation or migration of normally quiescent endothe~ cells in culture or promote neovascularization in vivo including factors which are associated with the vascularization that permits tumor growth; substances which are inhibitors of angiogenesis such as transforming growth factor ~, tumor necrosis factor ~, human platelet factor 4 (PF4~ and a interferon; substances which su~ ess cell migration, such as proteinA~e inh~hitors which inhibit proteases which may be necessary for penetration of the basement membrane, in particular, tissue inhibitors of metalloprot~nA~e TIMP-l and TIMP-2; and other proteins such as protamine which has demonstrated angiostatic properties. For a review of factors which play a role in angiogenesis see Maione T.E.
and R.J. Sharpe, TIPS, November 1990 Vol. ll page 457.
The transcriptional regulatory element of the invention may be u~ed in gene therapy to introduce a foreign gene into endotheliAl cells to ~u ~L or ~ev~lt vascular disoldc D- (See Nabel et al., JACC Vol 17, No.6, page 189B, 1991 for a discussion of gene transfer into vascular cells). For exsmple, the trsnscriptional ~ 94/~94 21 A 3 3 3 6 PCT/CA93/003~2 regulatory element of the invention may be used to express foreign genes at specific sites in the circulation.
Endothelial cells are found 8t diseased sites and accordingly, the transcriptio~l regulatory element of the invention may be used to target the a~euLic agents including antiroA~ulants, vasodil~tor, and angiogenic factors (see above discussion) to endo~heli~l cells found at disea~ed sltes. Thus, genetic modification of endothel; A 1 cellE utilizing the transcripti onA 1 regulatory element of the invention may be u~ed in the treatment of acquired vascular disorders such as hypertension, atherosclerosis restenosis, arthritis and cancer.
EndOthe1 ;A1 cells line all blood vessels and accordingly the transcriptional regulatory element of the invention may be used to target therapeutic agents into the bloodstream. Thus, genetic mo~ific~tion of endo~h~li A 1 cells utilizing the transcriptin~l regulatory element of the invention may al~o be used in the treatment of systemic or inherited disorders. For example, the transcripti O~A 1 regulatory element of the invention could be operatively l~nk~A to the factor VIII gene and introA~l~eA into a population of endoth~l ;A1 cells to correct a hemophili~ disorder.
Endo~h~ cells geneti~lly modified in vitro using the transcriptionAl regulatory element of the invention i.e transformant host cells or cell lines con~ining transformant host cells of the invention, may be used to deliver gene products to the vasculature. In particular, endotheli~l cells genetically modified in vitro using the transcriptional regulatory element of the invention may be introduced into the vascular wall by catheterization. Using this method, the a~euLic proteins may be h.L J~ e~ into diseased arterial segments. The method may be particularly useful for introducing growth inhibitor proteins into an angioplasty site in patients with restenosis who have undergone coronary angioplasty.
Endo~heli~l cells genetirAlly modified in vitro W094/~94 PCT/CA93/00352 -~4333 16 -using the transcriptional regulatory element of the in~ention may be used to improve the performance of prosthetic ~ascular grafts. Prosthetic vascular grafts may be seeded with endothPl;~l cells geneticAlly modified using the transcriptio~l regulatory element of the invention, to produce therapeutic proteins which may prevent thrombosis or promote le~o~ulation. Vascular stents may also be populated with genetically modified endoth~l ;A1 cells to reduce problem~ such 85 thrombosis.
A gene under the control of the transcriptional regulatory element of the invention i.e recombinant molecules of the invent~on, may b~ d~rectly introduced into endothel~Al cells in vivo using deli~ery vehicles such as retroviral vectors, ~denovir~l vectors and DNA
virus vectors. They may also b~ introduced into endothelial cells in vivo using phy~ical techniques such as microin~ection and elect O~G ~t$on or chemical methods such as ~o~ecipitation and incorporation of DNA into liposomes.
The invention will be more fully understood by reference to the following examples. However, these examples are merely inten~ to illustrate embodiments of the invention and are not to be construed to limit the scope of the invention.
RYaMPL~S
The following materials and methods were ut~li 7e~ in the investigations outli n~ in the examples:

AgR/J, DBA, and AgR/J x DBA recombinant inbred mouse DNA5 were obtA;n~ from Jackson Labs (Bar ~Arhor~
~aine), digested with AccI, blotted to Zeta-Probe nylon membrsne (Bio-Rad), and probed with the 1. 6 kb tek cDNA
lAh~lled by random priming (Feinh~rg~ A.P. & Vogelstein, B. (1983) Analyt. Biochem., 132, 6-13). Hybridization was performed overnight at 65- in 200 mM sodium phosphate pH7.0, 7% sodium dodecyl sulfate (SDS), 1~ bovine serum albumin (BSA), and 1 ~M EDTA. Pilters were washed twice at ~ 94/~94 2 1 4 3 3 3 ~ PCT/CA93/00352 55- in 2 x SSC (1 x SSC= 0.15 M NaCl, 0.015 N sodium citrate p~7.0) 2nd 0.1% SDS and twice in 0.2 x SSC and O.1% SDS, and exposed overnight to Rodak XAR-5 film.
Mice Embryos and adult mouse tissues were obt~i n~
from random bred CD-l stocks (Charles Ri~er, Quebec).
Embryos were staged as Day 0.5 on the morning of a vaginal plug.
~NA purification ~n~ ~n~ 1 y8; ~
Total RNA was extracted from pools of 30 to 40 Day 9.5 and 12.5 murine embryonic hearts with RNAzol (CINNA/BlOTECX Lab. Int.), with some added modifications.
Briefly, tissues were washed with ice cold phosphate buffered saline (PBS) and homogenized in 2.5 ml of RNAzol.
Chloroform (250 ~1) was added and the tubes were mixed vigorously and then chilled on ice for 15 min. The suspension was centrifuged for 15 min at 4' after which the aqueous pha~e was collected and re-extracted twice more with phenol/chloroform/i~oamyl ~l~ohol (25:24:1;
vol:vol:vol). The RNA was precipitated with an equal volume of isopropanol, collected by centrifugation, and the pellet resu~ 3e~ in diethylpyroc~rhonate (DEPC)-treated O.4 M sodium acetate, pH5.2. The RNA were then precipitated with two volumes of 95% ethanol, washed with 70% and 95% ethanol, dried, and resusp~n~ in DEPC
treated 0.3 M sodium acetate, pH5.2. The RNA
concentration was determined and the RNA stored at -70 until u~e.
Poly A - cont~inin~ RNA was purified from a pool of 100 to 150 Day 12.5 murine embryonic hesrts with a QuickPrep mRNA i~olation kit (Pharmacis) as outlined by the supplier.
- For Northern blot hybridization, 5 ~g of poly A
- cont~in;ng RNA from 12.5 day embryonic heart was electrophoresed through a formaldehyde-agarose gel and blotted to a Zeta-Probe nylon membrane (Bio-Rad) according to established protocols (Sambrook et al., 1989, Molecular ~ 43 PCT/CA93/00352 ~

Cloning. Cold Spring Harbor Laboratory Press). The membrane was hybridized with a [32P]-labelled antisense riboprobe synthQsized from the 1.6 kb tek cDNA in run off reactions with SP6 RNA polymerase (Promega).
Revarse Tr~nRcript-~on Coupled to the Polymerase ~h~i n Reaction (RT-PCR) First strand cDNA was synthesized in a total reaction volume of 20 ~l cont~inin~ 20 ~g of total RNA, 200 units of Mo-MLV-reverse transcriptase (BRL), either 1 ~g of oligo-d(T)18 (Day 12.5 RNA) (Ro~rhin~er M~nnh~m) or 2 ~g of r2ndom hexamer primers (Day 9.5 RNA) (Boerhinger M~nnh~im), 1 x PCR buffer (cetus)~ 2.5 mN MgCl2, 1 mM of dNTPs (Pharmaci~), 40 unit~ of RNAsin (Promega), and 12.5 mN dithiothreitol. The RNA was heated to 65-C for 10 min and cooled quickly on ice prior to addition to the reaction components. The reaction was allowed to proceed for l h ~t 37- and then terminated by hesting for 5 min at 95-. For PCR, the reaction mixture was ad~usted to a final volume of 100 ~l contAin~ng 1 x PCR buffer, 1.5 mM MgC12, 800 ~M dNTP~, ~nd 1 ~g of e~ch of the two degenerate tyrosine kinase ol1gorncleotide primers described by Wilks, A.F. (1989) Proc. Natl. Acad. Sci., 86, 1603-1607.
Amplification was performed with a Ericomp thermocycler using the following parameters: denaturation for 2 min at 94-, ~nn~Aling for 2 min at 42-, and extension for 4 min--at 63-. After 40 cycles, the reaction products were collected by ethanol precipitation and electrophoresed through at 2%
low-melt agarose (Sea Plaque) gel. In most cases a band of approximately 200 bp was visible within a h~c~-J-ound smear of e~hi~;nm bromide ~tA;n;n~. This band was excised and ecovered by three cycles of freeze-thaw in 100 ~l of water. 10 ~l of this solution was then sub~ected to a second round of PCR under the ~ame conditions described ~bove.
nin~ An~ ~equenc~n~ of RT-PCR ~roducts.
After the second round of amplification, 10 ~l of the reaction mixture were analyzed on a gel for ~ 094/~94 21433 PCT/CA93/00352 successful amplification. The remaining 90 ~1 were then ethanol precipitated, digested with EcoRI and BamHI, gel purified, and ligated to pGEM7Zf+ (Promega) digested with the same enzymes. The ligation mixture was then transformed into MVll90 competent cells, individual amp~
colonie~ picked, plasmid DNA prepared, and the cDNA
inserts analyzed by single track dideoxynucleotide sequencing (sanger~ F., Nicklen, S. & Coulson, A.R.
(1977). Proc. Natl . Acad. Sci ., 74, 5463-5467). A æingle representative clone of each multiple isolate was sequenced in its entirety. Of the 58 clones analyzed, roughly 10% howed no sequence identity to tyrosine kinases and were di~regarded.
Isolation of additional tçk cDNA sequences.
Approximately 106 plaques from an amplified, random primed 13.5 day murine embryonic AgtlO cDNA 1 ihr~ry were hybridized with the 210 bp tek PCR ~.odu-t l~h~l led with t32P]-dCTP by PCR. Hybridization wa8 carried out overni~ht at 55- in 50~ formamide, 10% dextran sulfate (Pharmaci~), 0.5 % BLOTTO, 4 x SSPE (1 x SSPE~ 0.18 ~
NaCl, 10 mM NaH~PO~, 1 m~ EDTA, pH7.4), 100 ~g/ml sheared salmon sperm DNA, and 2 x 106 cpm/ml of probe. Filters were washed at 55- twice in 2 x SSC contAinin~ 0.1% SDS and twice in 0.2 x SS~ contA~ning 0.1% SDS, dried, and exposed overnight to Kodak XAR-5 film. One clone was isolated from this screen and was found to contain a 1.6 kb cDNA.
The sequence of the 1.6 kb cDNA was determined by the method of Ssnger et al. (1977) from a set of anchored deletions generated with a stAn~rdized kit (Erase - A -Base, Promega).
Tn sitU h~yhri~iza~-ion Embryos isolated on Day 12.5 were dissected away from all extraembryonic tissue3 whereas embryos at earlier time points were ec~ d in utero. Embryos and adult tissues were fixed over~i~ht in 4% paraformaldehyde, dehydrated with A 1 cohol8 and xylenes, and emh~ in paraffin. Tissues were ~ectio~ at 6 ~m thickness and W094/~94 PCT/CA93/00352 ~
~4~ 20 -mounted on 3-aminopropyltriethoxysilane treated slides (sigma). After removal of paraffin the samples were treated with predigested pronase (Boerhinger M~nnheim), acetylated with triethanolamine, dehydrated, and hybridized according to the protocol described by Frohman, N.B., Boyle, M. & Martin, G.R. (1990), Development, 110, 589-607.
Dark and bright field photomicroscopy was performed with a Leitz Vario Orthomat 2 photomicroscopic system. Ad~acent section~ probed with a tek sense probe produced no detectable signal above background.
~ hole-mount in situ hybridizations were performed using a modification of existing procedures (Tautz, D. & Pfeifle, C. (1989). Chromosoma, 98,81-85;Hemmati-Brivanlou, A., Frsnck, D., Bolce, M.E., Brown, B.D., Sive, H.L. h Harland, R.M. (1990).
Development, 110, 325-330; Conlon and Rossant, in prep.).
The hybridization of single-stranded RNA probes l~helled with digoxigenin was detected with antidigoxigenin antiho~;es coupled to ~1~A1 in~ phosphatase. The En2 cDNA
was prepared as set forth in Joyner A.L. & Martin, G.R.
(1987). Genes and Dev., 1, 29-38 and expression of En2 i8 described in D~vi , C.A., Holmyard, D.P., Millen, R.J. &
Joyner, A.L. (1991) Development, 111:, 287-298.
Tnununohistochemistry Sections were st~n~ immllnnhi~tochemically for von Willebrand factor with a commercially svailable kit (Biomeda). After color development, slides were counterst~i n~ with Harris hematoxylin.
~ aMPT.R !
Isolation and chsrac~ri Z~t i on of te~
To identify and characterize tyrosine kinases expressed during murine cardiogenesis, cDNAs were synthesized from 9.5 and 12.5 day embryonic heart RNA by RT-PCR using degenerate oligonucleotide primers previously demonstrated to amplify tyrosine kinase sequences preferentially (Wilks, A.F. 1989, Proc.Natl.Acad.Sci., 86, ~ 094/~94 2 1 ~ 3 3 3 6 PCT/CA93/00352 1603-1607). Considerable cellular differentiation and morphogenesis have occurred within the cardiac region of the embryo by Day 9.5. At this stage the heart has developed from the primordisl mes~A~m cells of the cardiac plate into a primitive bent tube structure, consi~ting of two endoth~ l tubes enclosed within the developing myocardium. BeL-een Day 9.5 and 12.5 the heart undergoes additional complex morphological changes in association with the formation of the four chambers and septa characteristic of the adult heart. Sequence analysis of 58 clones obtAin~ following amplification revealed that whereas roughly 10% did not contain sequence similarities to protein kinases the rema~ nA~r correspon~ to 5 distinct cDNAs (Table 1 - Identity and number of tyrosine kina~e cDNA clones ecovered from Day 9.5 and 12.5 murine embryonic heart by RT-PCR). Four of these cDNAs represented previously characterized tyrosine kina~es including, bmk, c-src, c-abl, and the platelet derived growth factor receptor ~-~ubunit (pdgfrb). The isolation of bmk, c-src, and c-abl is consistent with the broad tissue distribution of these kinases (Wang, J.Y.J.
& Baltimore, D. (1983). Mol . Cell . Biol ., 3, 773-77g;
Ben-Neriah et al., (1986J. Cell, 44, 577-586; Holtzman, D., Cook, W. & Dunn, A. (1987). Proc.Natl.Acad.Sci., 84, 8325-8329; Ren~haw, M.W., Capozza, M.A. & Wang, J.~.J.
(1988). Mol.Cel~.Biol., 8, 4547-4551). The ec~vel~ from embryonic heart of pdgfrb at a relatively high frequency may i n~ icAte that pdgfrb plays an important role in cardiogene~is, as has been suggested by recent stl~ie~
demonstrating that the addition of PDGF-BB to explants of axolotol c~rdiac field mesoderm stimulates the production of beating bodies (Muslin, A.J. & Williams, L.T. (1991).
Development, 112, 1095-1101) the fifth cDNA, which was also isolated 8t high frequency, was novel and for reasons that will become clear below was designated tek . The 210 bp RT-PCR-derived tek clone wa~ subsequently used to isolate addit~ O~A 1 tek cDNA sequences-

4 PCr/CA93/00352 ~
2~4333~ - 22 Figure 2 shows the nucleotide sequence of a 1.6 kb tek cDNA isolated from a 13.5 day mouse embryo cDNA
library. Translation of this sequence reveals a single large open re~i ng fr_me that terminates with TAG at nucleotide 907, followed by 696 nucleotides of 3 untranslated sequence. Several features of the deduced amino acid sequence suggest that the 1.6 kb tek cDNA
qnC'OA~C the cytoplasmic portion of a transmembrane RTg, consisting of the catalytic domain followed by a short carboxy-terminal tail of 33 amino acid residue~s.
Figure 14 ~hows a comparison of the deduced amino acid sequence of tek with that of other tyrosine kinases; Identical sequences are denoted by periods.
r'A~h~s were added to allow for optimal alignment. The ki n~Re insert and conserved regions of the catalytic domain are in~il Ated beneath the aligned sequences (Hanks, S.R., Quinn, A.N. & Hunter, T. (1988), Science, 241, 52).
Comparative seq~evncc-s shown are for human Ret (TAkAh~hi, M. & Cov~el, G.M. (1987). ~ol.Cell.Biol., 7, 1378-1385), and Jtkl4 (Part~nen, J., M~ikelii, T.P., Alitalo, R., Lehv~laiho, H. & Alitalo, ~t. (1990) Proc.Natl.Acad.Sci., 87, 8913-8917) and murine Flg (Reid, H.H., Wilks, A.F. &
RernArd, O. (1990) Proc.Natl.Acad.Sc~ , 87, 1596-1600).
As shown in Figure 14, the putative kinase domain contains several sequence motifs conserved among tyrosine kinA~s~ including the tripeptide motif DFG, which is found in almost all known kinase~, and the consensus ATp-bin~ling site motifs c-x~;xx~i followed by A~R
16 amino acid residues downstream (Hanks et al., 1988).
Transmembrane RTR's possess a methio~ine residue wi~hin the motif ~M~T~ of conserved region VIII of the catalytic domain (Hanks et al., 1988) as does tek, and the catalytic domain is interrupted by a putative 21 amino acid kinA~e insert, a structural motif not found in cytoplasmic tyrosine kinases (Hanks et al., 1988).
Comparison with other tyrosine kinases (Figure 14) reveal~ that the A~ rc-~ tek amino acid sequence shows ~ 21 ~ 3 3 3 ~ PCT/CA93/00352 42% sequence identity to the mouse fibroblast growth factor receptor Flg (Reid et al., 1990; Safran, A., Avivi, A., Orr-Urtereger, A., Neufeld, G., Lonai, P., Givol, D.
& Yarden, Y. (1990). Oncogene, 5, 635-643, Sambrook, J., Fritsch, E.F. & ~aniatis, T. (1989). Molecular Cloning.
Cold Spring ~rhor Laboratory Press) and 45% to the transmembrane RTR en~o~ by the human c-ret proto-oncogene (T~kAhA~hi & Cooper, 1987). In sddition, striking ~equence identity is observed to a 65 amino acid residue sequence enro~ by Jtkl4, a putative tyrosine kinase cDNA isolated from differentiating human R562 cells by RT-PCR (Partanen et al., 1990). T~ken together, the results suggest that tek encodes a novel RTR.
Ry~Mpr.R TT
rhromosomal mappi n~ of the tek 1 OC"~
Napping of the tek locu~ was accomplished by monitoring the ~train distribution pattern of an AccI
restriction site polymorphism in recombinant inbred (RI) mouse strains derived from matings beL~een ARR/J (A) and DBA/2J (D) mice. The tek cDNA detects bands of 6.5, 6.1, 1.3 and 6.5, 3.1, 1.3 kb in DNA from the A and D ætrains, respectively. Southern blot hybridization analysis of DNA
from 24 RI mice with the 1.6 kb cDNA probe, and comparison of the segregation pattern with the Jackson Laboratory data base r revealed 95.8% cosegregation between tek and both brown and pmv-23, two loci that have previously been loc~li7ed to mouse chromosome 4 (Lyon & Searle, 1989).
Table 2 shows the co~egregation of the tek, brown, and pmv-23 loci in A x D strain~. In Tsble 2 for each RI strain, the 8ymbol shown indicates the presence of an allele characteristic of the progenitor from which the strain wa8 derived (A, ARR/J; D, DBA/2J) These data place tek between the brown and pmv-23 loci within 3.8+1.9 centimorgans of each interval.

wo 94,~4433~ 6 PCT/CA93/00352 ~

RYAMPLE III
Multiple tek-related transcripts are expressed i n e~bryonic heart tek expression in embryonic heart was examined by Northern blot hybridization using an antisense probe derived from the 1.6 kb tek cDNA. Figure 5 shows a Northern blot hybridization analysis of tek expression in 12.5 day murine embryonic heart; Arrows on the left denote the position of migration of 28 S and 18 S
ribosomal RNAs obt~ineA from ad~scent lane loaded with total RNA.
Figure 5 shows that the tek probe detects 4 transcripts of 4.5, 2.7, 2.2, and 0.8 kb in size in cardiac RNA from 12.5 day mouse embryos. These hybridizing ~pecie~ vary considerably in signal intensity, suggesting that they may differ in relative ab~n~nce, with expression of the 2.7 and 2.2 kb transcripts occu~ ing at significantly higher level~ than the 4.5 and O.8 kb RNAs. While the exact relationship among these transcripts is unclear, it i~ po~sible that they arise by differential splicing, since the 1.6 kb tek cD~A detects 8 single genomic locus in mouse DNA by Southern blot hybridization at the same stringency.
~YA~Pr-~ TV
In situ localization of tek expression during mouse embl yo~enes i ~
To determine which cell types express tek during development, RNA in situ hybridization analyses were performed on mouse embryos with an antisense riboprobe synthesized from the 1.6 kb tek cDNA.
Figure 6 shows the in situ hybridization analysis of tek expression in the 12.5 day embryo; A.
Dark field illumination of ~ para-sagittal section. Bars 600~m. B. and C. Bright and dark field illuminstion respectively, of the heart region taken from 8 mid-sagittal section. Bars 300 ~m. IV and VI~ fourth and sixth aortic arches; A, atrium: BA, basilar artery CV, caudal ~ 2143336 W094/~g4 ~ PCT/CA93/00352 vein; E, endocardium; L, liver; M, leptomeninges; ~a, mandible; My, myocardium; PC, pericardial cavity; RA, renal artery; SS, sino-auricular septum; SV, sinus venosus; V, ventricle.
Figure 6A shows that in 12.5 day mouse embryos, expression of tek i8 readily detected in the heart, the leptomeninge~ lining the brain and spinal cord, and the inner l~n~ng of ma~or blood ves~els, including the c~ Al vein and basilar and renal arteries. In addition, thin bands of hybridization are observed in the intersomite regions, correspo~i ng to tek expression in the intersegmental vessels. Close examination of the region of the developing heart (Figure 6B and 6C) reveals that tek i8 expre~ ed in the ~n~ocArdium, as well as in cells lining the lumina of the atria, the IV and VI aortic arches, the ~inus venosu~, and the ~ino-auricular septum.
In addition, tek expression is observed in numerous small blood ves~els perforating the liver and mandible. These observations, together with the overall pattern of hybridization seen in the 12.5 day embryo, demonstrate that tek i8 expressed in the endo~hel;Al cells of the tunica interna, the innermost lining of the blood vessels; hence the designation ~unica interna endothelial cell ~inase, tek.
More detailed information on tek expression-was obtAin~A through analysis of sections from earlier developmental stages. Hybridization to 6.5 and 7 day embryos revealed that while tek is expressed strongly in the inner lining of the small blood vessels and ~r~ llAries of the matern~l decidua, no expression is observed in either the embryo itself or the ectoplacental cone. The absence of tek expression at these stages is - consistent with the fact that at 6.5 to 7 days the embryo contains only a small amount of mesoderm from which endothel i A 1 cell~ are known to be derived.
Fi~ure 7 shows the expression of tek precedes that of von Willebrand factor in 8.5 day embryos;

W094/~94 PCT/CA93/00352 2 l 43 3 3 6 - 26 -Adjacent transverse sections through an 8.5 day embryo fixed in utero were either hybridized in situ with an t35S]-labelled tek probe or st~ine~ i~munohistochemically for von Willebrand factor. A. Bright field illumination of tek expression, Bar: 300 ~m. B. Dark field illumination of section in A. C. High magnification of a blood island, slightly out of the field shown in A, depicting silver grains over flat, elongated cells of endothelial-like morphology, Bar: 50 ~m. D. Ad~acent section to A at higher magnification showing absence of expression of von Willebrand factor in the embryo, 8ar:
100 ~m. E. Ad~acent section to A at higher magnification showing expression of von Nillebrand factor in the rldothPl ~ ning of the blood vessels of the matern~l decidua. Bar: 200 ~m. F. High magnifiration of-ceph~lic region in A 3howing silver grains over a large, round cell of angioblast-like morphology (arrow). B r: 50 ~m. G.
Bright field illumination of a sagittal ~ection of an 8 5 day embryo hybri~7~ in situ with an t35S]-l~h~lled tek probe. Bar: 300 ~m. H. Dark field illumination of G. I.
Higher magnification of heart region in A showing silver grains over cell with endoth~ nd angioblast-like morphology in the developing en~oc~dium. Bar: 100 ~m.
J. Higher magnification of somite region in A showing tek-expressing cells exten~ing beneath, and possibly from, the vel~L.al surface of the somites. B~r: 100 ~m. A, amnion;
Ag, presumptive angioblast; BI, blood island; D, maternal decidua; DA, dorsal aorta; E, ~nAoc~dium; Ec, ectoplacental cone; En, endoth~ l cell; G, foregut; HV, head vein; NF, neural fold; S, somite; Y, yolk sac.
RNA in situ analysis of 8.0 day embryos revealed that tek e~ession fir~t becomes detectable in the developing yolk ~ac and ~ few small clusters of cells in the cerhAlic mesenchyme. Thi~ expression becomes more ~o..o~.cad by Day 8.5, at which time significant hybridization can be observed in the mesodermal component of the amnion (outer cell layer) and yolk sac (inner cell ~ 21 4 3 3 3 6 PCT/CA93/00352 layer), as well a~ in the developing en~o~rdium and the inner 1 ;n;ng of the head veins and dorsal aortae (Figure 7A and 7B). In addition, sagittal sections reveal numerous focal areas of hybridization throughout the cephalic mesenchyme in regions thought to contain developing vasculature, a8 well as a small number of tek-expressing cells ext~n~ing beneath the ventral surface of the somites (Figure 7H and 7J).
Whole mount in situ hybridization analysi~
confirmed and ex~en~ the above observations, as well as provided a three dimensional ~e s~e~Live on tek expression during embryogenesi~. Figure 8 shows tek expression in whole mount embryos; A., B., C. and D. tek expression in Day 8.0 embryos. E. tek mRNA distribution in a Day 9.5 embryo. F. En2 e~ression in a Day 8 embryo. I, II, III, first, second and third aortic arches; DA. dorsal aorta;
E, endocardium; G, foregut pocket; H, heart; IS, intersegmental v~sel; Ny, myocardium; ; NF, neural fold;
OT; otic vesicle; V, vi~ell in~ vein; Y, yolk sac. Bars:
250 ~m.
Consistent with our observations with sectioned material, localized tek expression was not observed on embryonic Day 7. The first detectable expression was seen about the time of first ~omite formation when signal was observed in the yolk ~ac, head mesenchyme, and heart. In Day 8. 5 embryos, tek was found to be expressed in these same areas, and in the paired dorsal aortae, the vitelline veins, and in the forming intersegmental vessels (Figure 8). By this time, tek expression was clearly confined to blood vessels within the embryo. On Day 9, tek expression was ~een in addition, in the aortic arches and expre~sion was very striking in the en~sc~rdium (Figure 8E). Control hybridizations with an En-2 probe demonstrated the specificity of tek RNA detection (Figure 8F).
LE V
pression of tek in endotheli~l c~ll progenitors The observation that tek is expre~sed beL~een WO94/~94 PCT/CA93/00352 ~
~4333~ 28 -Day 8.0 and 8.5 in focal regions thought to represent developing blood vessels raised the possibility that tek might be expresced in endoth~ cell progenitors.
Tn~ee~ close inspection of hybridized sections from 8 to 8.5 day embryos revealed that while the expression of tek in the matsrn~l decidua is restricted to cells of an endoth~ l cell morphology, tek expressing cells in the embryo are of two morphologically distinct cell types. In the developing blood islands of the yolk sac, where tek expression is first detected, silver grains are localized predominantly to elongated cells with characteri~tic endo~heli~l cell morphology (Figure 7C). In contrast, within the reph~lic me~enchyme, silver grains are frequently observed over large, round cells that, on the basis of similar morphology to cells described during avian embryogenesis (Par~n~tl~ et al., 1987; Coffin &
Poole, 1988; Noden, 1989; ~'oAen, 1991) ~ correspond to angioblasts, the presumptive progenitor of endoth~l~Al cells (Figure 7F). Both cell types are observed in the developing en~ocArdium (Figure 7I) which, at later stages, is known to contain only fully mature endothelial cells.
To characterize more precisely the staging of tek expression within the endothelial lineage, sections ad~acent to those u3ed for in situ hybridization were st~i n~A immunohictochemically for von Willebrand factor, a well characterized marker of mature endo~hsli~l cells (Jaffe, E.A., Hoyer, L.W. & Nachman, R.L. (1973).
J . Clin . Invest ., 52, 2757-2764; Hormia, M., Lehto, V.-P. &
Virtanen, I. (1984), Eur.J.Cell.Biol., 33, 217-228).
Figure 7B and H shows that whereas tek is expressed in both the maternal decidua and the embryo at Day 8.5, expression of von ~illebrand factor is observed only in the tek-expressing, vascular endotheli~l cells of the maternal decidua (Figure 7D and 7E). Hence tek expression eced~s that of von Nillebrand factor during embryogenesis. The same scenario is observed at later developmental stage8 during vascularization of individual 21 ~ 3 3 3 ~ PCT/CA93/00352 organs.
Figure 9 shows the expression of te~ precedes that of von Willebrand factor in the developing leptomeninges, A. Absence of immunohistochemical StA jn;ng of von Willebrand factor in Day 12.5 leptomeninges. Arrow denotes a large blood vessel faintly positive for von Willebrand factor. B. In situ detection of tek expression in Day 12.5 leptomeninges. C. St~i n ~ ng of von Willebrand factor in Day 14.5 leptomeninges. Day 14.5 leptomening~s were positive for tek expression (not shown). M, leptomeninges. Bars: 200 ~m.
Figure 9 shows that in the 12.5 day em~hryo, the developing leptomeninges hybridizes strongly with tek but fails to st~in positive for von Nillebrand factor. By Day 14.5, however, expression of von Willebrand factor can be readily detected in the leptomeninges. Assuming that there i8 not a 8ign~ f~CAnt lag ~eL~_en transcription and translation of von Willebrand factor, these observations, together with those on the morphology of tek-expre~sing cell8 ~ suggest that tek i~ expressed in both mature endoth~l~Al cells and their ~oye~itors.
~MPr.~ VT
tek is expressed in adult vasculature While the above results establish that tek i8 expressed during vascularization of the embryo, it was also of interest to determine whether expreæsion of tek is maint~in~ in endothelial cells of the adult. In situ hybridization analysis of a ~ection through the heart region of a 3 week-old mouse revealed that tek is expressed in the endocardium as well as in the ~ h~ l 1 ining of ma~or blood vessels, both arteries and veins, ronn~cting with the adult heart (Figure 10).
Figure lO shows the expression of tek in adult vasculature. A. Bright field illumination of a section through the upper heart region of a 3 week-old mouse hybridized with ~n t35S]_lAh~lled tek probe. Bars 20 ~m.
B. and C.

wo 94/~33~ ~ PCT/CA93/00352 -Bright field illumination showing tek expression in endoth~l iAl cells lining the artery and vein respectively. Bar: 1 ~m. Immnnshi~tochemical st~ining Of ad~acent sections revealed that,structures positive for tek expression also st~inPA positive for von Willebrand factor. A, artery; Bl, extravà~ated blood; T, trachea; V, vein.).
The intensity of the hybridization signal observed for these structures is considerably lower than that observed for the en~sc~rdium and blood vessels of 12.5 day embryos hybridized and ~oc~ed in parallel.
This could i n~ i cAte that mature endothelial cells, which are thought to be resting, have a different quantitative or qualitative requirement for e~ r~ion of tek.
l~Al~Pr.R VTT
De~r~in~t-ion of the ini~ ion s~te of tr~n~lation of tek Additin~Al cDNA sequences spAnn i ng the entire tek cDNA were obtA ~ n~ by screen~ n~ cDNA libraries using well establi~h~ protocols (Sambrook et al, 1989, Molecular Cloning, A Laboratory ~n~ Cold Spring Harbor-Lab. Press). Analysis of the complete cDNA sequence allowed determination of the most probable ~tart of translation for the following reasons: (1) The putative initiation codon (Methionine) is followed by a stretch of 23 amino acid~ which are of sufficient Lyd~OphObicity that they could serve as a signal peptide. (2) The re~ng frame does not contain any stop codons for 1118 amino acids and the deri~ed amino acid sequence contains prLmary seguence motifs that are characteristic of receptor tyrosine kinases. (3) the other two forward r~;ng frames are not open for any significant distance and contain multiple ~top ~o~n~.
RYA~lPr.R yTTI .
A DNA segment was isolated from a mouse genomic bacteriophage library using a 5'-prime probe, consisting of nucleotides 0 to 912 of the tek cDNA, which cont~n~
the initiation codon and untranslated sequences of tek ~ 214 3 3 3 6 PCT/CA93/00352 using the procedures of Sambrook et al., 1989, Nolecular Cloning, A Laboratory ~nll~l. Cold Spring Harbour Lab.
Press. The DNA segment was cloned in the plasmid pGEm72F' and propagated in E. coli K12. A 16 kb phage clone was shown by hybridization with this 5~-prime probe and ~equence analysis using oligonucleotides specific for the cDNA sequence and the plasmid backbone, to contain a single exon with 175 bp homologous to the cDNA. A DNA
fragment exten~ ng from the Bgl II restriction site located at nucleotide 110 of the cDNA to the nearest ~pn I in the phage was cloned upstream of the bacterial gene lacZ. This reporter construct contAini~g 7.2 kb of the tek gene was microin~ected into pronuclei of fertilized randomly bred CD-l mice using procedures as set out in Hogan et al., 1986, M~nir~l~ting the Mouse Embryo, A
Laboratory M~n~ . Cold Spring ~rhor Lab. Press. Three transgenic founder embryos were dissected from their foster mothers and yolk æac DNA analyzed for the presence of the transgene. Expression of the transgene was determined by the X-gal st~i n i ng of whole embryos and subseguent sec~ioni ng of the embryos. Figure 12 shows expression of LacZ in Day 8.5 embryos and Figure 13 shows mRNA distribution in a Day 8.5 embryo. The 7.2 kb fragment was able to drive lacZ expression in endothelial cells that had previously been shown to express tek RNA
(Figure 12) thus demonstrating that this DNA cont~in~ the tek promoter.
-333~

Table 1: Protein tyrosine Wnase cDNAs ~o'- ed by RT PCR

Embryonic Age (Days) cDNA
DSb cabl c-s~r bmk 9.S 26 7 2 12.5 S 10 - - -21~3336 ~o 94/04694 Pcr/cA93~003s2 TAR~.F. 2. Cosc~g&llo~ Or the ~ck, b~, and pmv-2~ locl in A ~ D stralns.

A x D strai~
Locus 1 2 3 6 7 8 9 10111213141S1618202122232425262728 tek D D A D D A A A D A D A D D D D A D D A D D D D
btown D D A D D A A A D A A A D D D D A D A A D D D D
pmv-23 D D A D D A D A D A D D D A D D A D D A D D D A

WO 94/ ~ 94 PCT/CA93/00352 -~ 4333~ 34 -SEQ~NC~ LISTING
(1) GENERAL INFORMATIONs (i) APPLICANTs Mount Sinai Hospital Corporation Breitman, Nartin L
Dumont, Daniel J.
~r Gradwohl, Gerard G ..
(ii) TITLE OF lNv~.LlONs Tissue Specific Transcriptional - Regulatory Element (iii) NuM~n OF SE~U~NC~Ss 5 (iv) CO~R~SPONDENCE ADDRESSs 'A~ AnDR~S~S~s Linda M. Kurdydyk, Bereskin & Parr B STREET: Box 401, 40 Ring Street West C CITYs Toronto D STATE: Ontario E COUNTRY: CAnA~A
~F, ZIP: N5H 3Y2 (v) COM~u~n R~An~RT.~ FORM:
'A~ MEDIUM TYPEs Floppy disk B CO..~u~n: IBN PC compatible C OPERATING SY~L~IIs PC-DOS/MS-DOS
D, SOFTWARE: PatentIn Release #1.O, Ver~on #1.25 (vi) CuRR~NT APPLICATION DATA:
'A' APPLICATION NW~KS
B FILING DATEs 25-AUG-1993 ~C CLASSIFICATIONs (viii) ATTORNEY/AGENT INFORMATION:
'A' NANEs Kurdydyk, Linds M
B REGISTRATION NW~RS 34,971 ~R~Nr~/DGc~L Nu~ ns 3153-086/LNK
(ix) TRT.~ uNlCATIoN INFORNATIONs 'A' TEL~nOh~: (416) 364-7311 B TELEFAXs (416) 361-1398 ~C~ TELEXs 06-23115 (2) lN ~O~ ~TION FOR SEQ ID NO:l:
(i) S~UU~NC~ CHARACTERISTICS:
'A' LENGTH: 4176 base pairs B TYPEs nucleic acid C STRAND~nN~SSs single D~ TOPOLOGYs l~n~Ar ( ii ) MnT-~r,~TT-~ TYPE: cDNA
= (iii) nY~OL.~-LlCAL: NO

~ og4/~94 21 4 3 3 3 G PCT/CA93/00352 (iv) ANTI-SENSE: NO
(v) F~ A~M~T TYPE: N-terminal (vi) ORIGINAL SOURCE:
(A) ORGANISM: ~us musculus 'B) STRAIN: CD-l D) DEVELOPNENTAL STAGE: Embryo ,F) TISSUE TYPE: Heart (vii) IMMEDIATE SOURCE:
(B) CLONE: tek (viii) POSITION IN GENOME:
(A) C~ROMOISOME/SEGNENT: 4 (B) MAP POSITION: Between the brown and pmv-23 loci (ix) FEATURE:
! A) NAME/REY: CDS
B) LOCATION: 124..3489 ~D) OTHER INFORMATION: /function= "putative transmembrane receptor"
/product= "tyrosine kinase"
/gene= "tek"
/st~n~rd_name="proteintyrosinekinasereceptor"

(xi) SEQU~N~ n~SrRTPTION: SEQ ID NO:l:
GCCAACTTGT AAACAAGAGC GAGTGGACCA TGCGAGCGGG AAGTCG~AAA ~ ~AGTT

GTTGAAAGCT Tccr~Gr~5Ac TCATGCTCAT CTGTGGACGC TGGATGGGGA GATCTGGGGA

AGT ATG GAC TCT TTA GCC GGC TTA GTT CTC TGT GGA GTC AGC TTG CTC

Met Asp Ser Leu Ala Gly Leu Val Leu Cys Gly Val Ser Leu Leu CTT TAT GGA GTA GTA GAA GGC GCC ATG GAC CTG ATC TTG ATC AAT TCC

Leu Tyr Gly Val Val Glu Gly Ala Met Asp Leu Ile Leu Ile Asn Ser CTA CCT CTT GTG TCT GAT GCC GAA ACA TCC CTC ACC TGC ATT GCC TCT

Leu Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys Ile Ala Ser GGG TGG CAC CCC CAT GAG CCC ATC ACC ATA GGA AGG GAC TTT GAA GCC

Gly Trp His Pro His Glu Pro Ile Thr Ile Gly Arg Asp Phe Glu Ala W094/~K94 PCT/CA93/003~2 ~1~333~

TTA ATG AAC CAG CAC CAA GAT CCA CTG GAG GTT ACT CAA GAT GTG ACC

Leu Met Asn Gln His Gln Asp Pro Leu Glu Val Thr Gln Asp Val Thr AGA GAA TGG GCG AAA AAA GTT GTT TGG AAG AGA GAA AAG GCC AGT AAG

Arg Glu Trp Ala Lys Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys ATT AAT GGT GCT TAT TTC TGT GAA GGT CGA GTT CGA GGA CAG GCT ATA
456le Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Arg Gly Gln Ala Ile AGG ATA CGG ACC ATG AAG ATG CGT CAA CAA GCA TCC TTC CTA CCT GCT
504rg Ile Arg Thr Met Lys Met Arg Gln Gln Als Ser Phe Leu Pro Ala ACT TTA ACT ATG ACC GTG GAC AGG GGA GAT AAT GTG AAC ATA TCT TTC

Thr Leu Thr Met Thr Val Asp Arg Gly Asp Asn Val Asn Ile Ser Phe AAA AAG GTG TTA ATT AAA GAA GAA GAT GCA GTG ATT TAC AAA AAT GGC

Lys Lys Val Leu Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn Gly TCC TTC ATC CAC TCA GTG CCC CGG CAT GAA GTA CCT GAT ATT TTA GAA

Ser Phe Ile His Ser Val Pro Arg His Glu Val Pro Asp Ile Leu Glu GTT CAC TTG CCG CAT GCT CAG CCC CAG GAT GCT GGT GTG TAC TCG GCC
696al His Leu Pro His Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser Ala AGG TAC ATA GGA GGA AAC CTG TTC ACC TCA GCC TTC ACC AGG CTG ATT
744rg Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg Leu Ile GTT CGG AGA TGT GAA GCT CAG AAG TGG GGG CCC GAC TGT AGC CGT CCT

Val Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro Asp Cys Ser Arg Pro TGT ACT ACT TGC AAG AAC AAT GGA GTC TGC CAT GAA GAT ACC GGG GAA

Cys Thr Thr Cys Lys Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu 21~ 3 3 3 6 PCr/CA93/00352 -- 37 _ TGC ATT TGC CCT CCT GGG TTT ATG GGG AGA ACA TGT GAG AAA GCT TGT

Cys Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys GAG CCG CAC ACA TTT GGC AGG ACC TGT AAA GAA AGG TGT AGT GGA CCA
936lu Pro His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Pro GAA GGA TGC AAG TCT TAT GTG TTC TGT CTC CCA GAC CCT TAC GGG TGT
984lu Gly Cys Lys Ser Tyr Vsl Phe Cys Leu Pro Asp Pro Tyr Gly Cys TCC TGT GCC ACA GGC TGG AGG GGG TTG CAG TGC AAT GAA GCA TGC CCA

Ser Cys Ala Thr Gly Trp Arg Gly Leu Gln Cyfi Asn Glu Ala Cys Pro TCT GGT TAC TAC GGA CCA GAC TGT AAG CTC AGG TGC CAC TGT ACC AAT

Ser Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Arg Cy~ Nis Cys Thr Asn GAA GAG ATA TGT GAT CGG TTC CAA GGA TGC CTC TGC TCT CAA GGA TGG

Glu Glu Ile Cys Asp Arg Phe Gln Gly Cys Leu Cys Ser Gln Gly Trp CAA GGG CTG CAG TGT GAG AAA GAA GGC AGG CCA AGG ATG ACT CCA CAG
1176ln Gly Leu Gln Cys Glu Lys Glu Gly Arg Pro Arg Net Thr Pro Gln ATA GAG GAT TTG CCA GAT CAC ATT GAA GTA AAC AGT GGA AAA TTT AAC
1224le Glu Asp Leu Pro Asp His Ile Glu Val Asn Ser Gly Lys Phe Asn CCC ATC TGC AAA GCC TCT GGG TGG CCA CTA CCT ACT AGT GAA GAA ATG

Pro Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Ser Glu Glu Met ACC CTA GTG AAG CCA GAT GGG ACA GTG CTC CAA CCA AAT GAC TTC AAC

Thr Leu Val Lys Pro Asp Gly Thr Val Leu Gln Pro Asn Asp Phe Asn TAT ACA GAT CGT TTC TCA GTG GCC ATA TTC ACT GTC AAC CGA GTC TTA

Tyr Thr Asp Arg Phe Ser Val Ala Ile Phe Thr Val Asn Arg Val Leu WO94/~K94 PCT/CA93/003~2 -2~4333&
.

CCT CCT GAC TCA GGA GTC TGG GTC TGC AGT GTG AAC ACA GTG GCT GGG
1416ro Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly ATG GTG GAA AAG CCT TTC AAC ATT TCC GTC AAA GTT CTT CCA GAG CCC
1464et Val Glu Lys Pro Phe Aæn Ile Ser Val Lys Val Leu Pro Glu Pro CTG CAC GCC CCA AAT GTG ATT GAC ACT GGA CAT AAC TTT GCT ATC ATC

Leu His Ala Pro Asn Val Ile Asp Thr Gly His Asn Phe Ala Ile Ile AAT ATC AGC TCT GAG CCT TAC TTT GGG GAT GGA CCC ATC AAA TCC AAG

Asn Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro Ile Lys Ser Lys AAG CTT TTC TAT AAA CCT GTC AAT CAG GCC TGG AAA TAC ATT GAA GTG

Lys Leu Phe Tyr Lys Pro Val Asn Gln Ala Trp Lys Tyr Ile Glu Val ACG AAT GAG ATT TTC ACT CTC AAC TAC TTG GAG CCG CGG ACT GAC TAC
1656hr Asn Glu Ile Phe Thr Leu Asn Tyr Leu Glu Pro Arg Thr Asp Tyr GAG CTG TGT GTG CAG CTG GCC CGT CCT GGA GAG GGT GGA GAA GGG CAT
1704lu Leu Cys Val Gln Leu Ala Arg Pro Gly Glu Gly Gly Glu Gly His CCT GGG CCT GTG AGA CGA TTT ACA ACA GCG TGT ATC GGA CTC CCT CCT

Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Cys Ile Gly Leu Pro Pro CCA AGA GGT CTC AGT CTC CTG CCA AAA AGC CAG ACA GCT CTA AAT TTG

Pro Arg Gly Leu Ser Leu Leu Pro Lys Ser Gln Thr Ala Leu Asn Leu ACT TGG CAA CGG ATA TTT ACA AAC TCA GAA GAT GAA TTT TAT GTG GAA

Thr Trp Gln Pro Ile Phe Thr Asn Ser Glu Asp Glu Phe Tyr Val Glu GTC GAG AGG CGA TCC CTG CAA ACA ACA AGT GAT CAG CAG AAC ATC AAA

Val Glu Arg Arg Ser Leu Gln Thr Thr Ser Asp Gln Gln Asn Ile Lys ~ 094/~94 214 3 3 3 6 PCT/CA93/003~2 GTG CCT GGG AAC CTG ACC TCG GTG CTA CTG AGC AAC TTA GTC CCC AGG

Val Pro Gly Asn Leu Thr Ser Val Leu Leu Ser Asn Leu Val Pro Arg GAG CAG TAC ACA GTC CGA GCT AGA GTC AAC ACC AAG GCG CAG GGG GAG

Glu Gln Tyr Thr Val Arg Ala Arg Val Asn Thr Lys Ala Gln Gly Glu TGG AGT GAA GAA CTC AGG GCC TGG ACC CTT AGT GAC ATT CTC CCT CCT

Trp Ser Glu Glu Leu Arg Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro CAA CCA GAA AAC ATC AAG ATC TCC AAC ATC ACT GAC TCC ACA GCT ATG

Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr Asp Ser Thr Ala Met GTT TCT TGG ACA ATA GTG G~T GGC TAT TCG ATT TCT TCC ATC ATC ATC

Val Ser Trp Thr Ile Val Asp Gly Tyr Ser Ile Ser Ser Ile Ile Ile CGG TAT AAG GTT CAG GGC AAA AAT GAA GAC CAG CAC ATT GAT GTG AAG

Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His Ile Asp Val Lys ATC AAG AAT GCT ACC GTT ACT CAG TAC CAG CTC AAG GGC CTA GAG CCA

Ile Lys Asn Ala Thr Val Thr Gln Tyr Gln Leu Lys Gly ~eu Glu Pro GAG ACT ACA TAC CAT GTG GAT ATT TTT GCT GAG AAC AAC ATA GGA TCA

Glu Thr Thr Tyr His Val Asp Ile Phe Ala Glu Asn Asn Ile Gly Ser AGC AAC CCA GCC TTT TCT CAT GAA CTG AGG ACG CTT CCA CAT TCC CCA

Ser Asn Pro Ala Phe Ser His Glu Leu Arg Thr Leu Pro His Ser Pro GGC TCT GCA GAC CTC GGA GGG GGA AAG ATG CTA CTC ATA GCC ATC CTT

Gly Ser Ala Asp Leu Gly Gly Gly Lys Met Leu Leu Ile Ala Ile Leu ~ 740 745 750 GGG TCG GCT GGA ATG ACT TGC ATC ACC GTG CTG TTG GCG TTT CTG ATT

Gly Ser Ala Gly Met Thr Cys Ile Thr Val Leu Leu Ala Phe Leu Ile wo 94/4~ 3 6 PCr/CA93/0035~ -ATG TTG CAA CTG AAG AGA GCA AAT GTC CAA AGG AGA ATG GCT CAG GCA

Net Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Arg,Met Ala Gln Ala TTC CAG AAC AGA GAA GAA CCA GCT GTG CAG TTT AAC TCA GGA ACT CTG

Phe Gln Asn Arg Glu Glu Pro Ala Val Gln Phe Asn Ser Gly Thr Leu GCC CTT AAC AGG AAG GCC AAA AAC AAT CCA GAT CCC ACA ATT TAT CCT

Ala Leu Asn Arg Lys Ala Lys Asn Asn Pro Asp Pro Thr Ile Tyr Pro GTG CTT GAC TGG AAT GAC ATC AAG TTT CAA GAC GTG ATC GGA GAG GGC

Val Leu Asp Trp Asn Asp Ile Lys Phe Gln Asp Val Ile Gly Glu Gly . 820 825 830 AAC TTT GGC CAG GTT CTG AAG GCA CGC ATC AAG AAG GAT GGG TTA CGG
2664sn Phe Gly Gln Val Leu Lys Ala Arg Ile Lys Lys Asp Gly Leu Arg ATG GAT GCC GCC ATC AAG AGG ATG AAA GAG TAT GCC TCC AAA GAT GAT

Met Asp Ala Ala Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp Asp CAC AGG GAC TTC GCA GGA GAA CTG GAG GTT CTT TGT AAA CTT GGA CAC

His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly His CAT CCA AAC ATC ATT AAT CTC TTG GGA GCA TGT GAA CAC CGA GGC TAT

His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly Tyr TTG TAC CTA GCT ATT GAG TAT GCC CCG CAT GGA AAC CTC CTG GAC TTC
2856eu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly Asn Leu Leu Asp Phe goo 905 910 CTG CGT AAG AGC AGA GTG CTA GAG ACA GAC CCT GCT TTT GCC ATC GCC
2904eu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Ile Ala AAC AGT ACA GCT TCC ACA CTG TCC TCC CAA CAG CTT CTT CAT TTT GCT

Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Leu Leu His Phe Ala ~g4 21 ~ 333 6 PCI~/CA93/00352 GCA GAT GTG GCC CGG GGG ATG GAC TAC TTG AGC CAG AAA CAG TTT ATC

Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gln Lys Gln Phe Ile CAC AGG GAC CTG GCT GCC AGA AAC ATT TTA GTT GGT GAA AAC TAC ATA

His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr Ile 960 9~;5 970 975 GCC AAA ATA GCA GAT TTT GGA TTG TCA CGA GGT CAA GAA GTG TAT GTG

Ala Lys Ile Ala Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr Val AAA AAG ACA ATG GGA AGG CTC CCA GTG CGT TGG ATG GCA ATC GAA TCA

Lys Lys Thr Net Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu Ser CTG AAC TAT AGT GTC TAT ACA ACC AAC AGT GAT GTC TGG TCC TAT GGT

Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly GTA TTG CTC TGG GAG ATT GTT AGC TTA GGA GGC ACC CCC TAC TGC GGC

Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Thr Pro Tyr Cy5 Gly ATG ACG TGC GCG GAG CTC TAT GAG AAG CTA CCC CAG GGC TAC AGG CTG

Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Leu GAG AAG CCC CTG AAC TGT GAT GAT GAG GTG TAT GAT CTA ATG AGA CAG

Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr Asp Leu Net Arg Gln TGC TGG AGG GAG AAG CCT TAT GAG AGA CCA TCA TTT GCC CAG ATA TTG

Cy8 Trp Arg Glu Lys Pro Tyr Glu Arg Pro Ser Phe Ala Gln Ile Leu GTG TCC TTA AAC AGG ATG CTG GAA GAA CGG AAG ACA TAC GTG AAC ACC

Val Ser Leu Asn Arg Net Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr ACA CTG TAT GAG AAG TTT ACC TAT GCA GGA ATT GAC TGC TCT GCG GAA

Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly Ile Asp Cys Ser Ala Glu WO94/~K94 PCT/CA93/00352 -2'14333~ - ~2 -GAA GCA GCC TAGAGrAr~AA CTCTTCATGT ACAACGGCCA TTTCTCCTCA

Glu Ala Ala CTGGCGCGAG AGCCTTGACA ccTG~AcrAA GrAAr-rr-Arc CACTGCCAAG AGATGTGATA

TATAAGTGTA TATATTGTGC lGT~l~l~GG ACCCTCCTCA TACAGCTCGT GCGGATCTGC

A~ Gl-~Cl GACTCTAATG TGACTGTATA TACTGCTCGG AGTAAr.AATG TGC~AA~A~C

AGAATGCCTG ll~lGGTTT rA~ATAA~A~ ALLl~lClAA AAGrA~A~-AT TGCACAGGAA

GGTATGAGTA ~A~ATACTGT AATGCATAAC ~ lATTGT CCTAGATGTG TTTGACATTT

TTCCTTTACA ACTGAATGCT ATAAAAGTGT TTTGClG-l~l GCGCGTAAGA TA~ CGT

ATTCCCTTGA cAGrAcAG~ A~AAAAGCGA ~ AAATGTA TGGATTATAT

TAAATGTGGG TTAC~ACA~ A~AGr7cr-~AA CATTCCAAGT AGrAr-AAGAr~ AGG~lcl~lC

AACTCTGCTC CTCACCTGCA GAAGCCAGTT ~ ~GCCA TGTr~Ac~A~T GTC~

TTTATAGCAC Cr-AAATCATT CTAAAA~A~G AACATCTAAA AACTTTGCTA GGAGACTAAG

AACCTTTGGA ~A~.A~Ar.A~A TAAGTACGGT CAAAAAAcA~ AACTGCG

(2) lN ~O~NATION FOR SEQ ID NO: 2:
(i) SEQ~Nc~ ~ARArTERISTICS:
'A) LENGTH: 1122 amino acid~
B) TYPE: amino acid ~D) TOPOLOGY: li ne~r (ii) MOLECULE TYPE: protein (xi) SEQu~NC~ DESCRIPTION: SEQ ID NO:2:
Met Asp Ser Leu Ala Gly Leu Val Leu Cys Gly Val Ser Leu Leu Leu Tyr Gly Val Val Glu Gly Ala Met Asp Leu Ile Leu Ile Asn Ser Leu 94/~94 21~ 3~3 6 PCI'/CA93/00352 Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys Ile Ala Ser Gly Trp His Pro His Glu Pro Ile Thr Ile Gly Arg Asp Phe Glu Ala Leu Met Asn Gln His Gln Asp Pro Leu Glu Val Thr Gln Asp Val Thr Arg lu Trp Ala Lys Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys Ile sn Gly Ala Tyr Phe Cys Glu Gly Arg Val Arg Gly Gln Ala Ile Arg Ile Arg Thr Met Lys Met Arg Gln Gln Ala Ser Phe Leu Pro Ala Thr Leu Thr Met Thr Val Asp Arg Gly Asp Asn Val Asn Ile Ser Phe Lys I.ys Val Leu Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser he Ile His Ser Val Pro Arg His Glu Val Pro Asp Ile Leu Glu Val is Leu Pro His Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser Ala ArSJ

Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg Leu Ile Val Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro Asp Cy8 Ser Arg Pro Cy8 Thr Thr Cys Lys Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys le Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu ro His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Pro Glu Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser Cys Ala Thr Gly Trp Arg Gly Leu Gln Cy8 Asn Glu Ala Cy8 Pro Ser Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Arg Cys His Cys Thr Asn Glu W094/046g4 333~ PCI/CA93/0035t ---- 44 _ Glu Ile Cys Asp Arg Phe Gln Gly Cys Leu Cys Ser Gln Gly Trp Gln ly Leu Gln Cys Glu Lys Glu Gly Arg Pro Arg Met Thr Pro Gln I le Glu Asp Leu Pro Asp His Ile Glu Val Asn Ser Gly Lys Phe Asn Pro Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Ser Glu Glu Met Thr Leu Val Lys Pro Asp Gly Thr Val Leu Gln Pro Asn Asp Phe Asn Tyr hr Asp Arg Phe Ser Val Ala Ile Phe Thr Val Asn Arg Val Leu Pro ro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met Val Glu Lys Pro Phe Asn Ile Ser Val Lys Val Leu Pro Glu Pro Leu 435 440 ~45 His Ala Pro Asn Val Ile Asp Thr Gly His Asn Phe Ala Ile Ile Asn Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro Ile Lys Ser Lys Lys eu Phe Tyr Lys Pro Val Asn Gln Ala Trp Lys Tyr Ile Glu Val Thr sn Glu Ile Phe Thr Leu Asn Tyr Leu Glu Pro Arg Thr Asp Tyr Glu eu Cys Val Gln Leu Ala Arg Pro Gly Glu Gly Gly Glu Gly His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Cys Ile Gly Leu Pro Pro Pro Arg Gly Leu Ser Leu Leu Pro Lys Ser Gln Thr Ala Leu Asn Leu Thr rp Gln Pro Ile Phe Thr Asn Ser Glu Asp Glu Phe Tyr Val Glu Val lu Arg Arg Ser Leu Gln Thr Thr Ser Asp Gln Gln Asn Ile Lys Val ro Gly Asn Leu Thr Ser Val Leu Leu Ser Asn Leu Val Pro Arg Glu ~O 94/04694 21 ~ 3 3 3 6 PCI /CA93/003~2 Gln Tyr Thr Val Arg Ala Arg Val Asn Thr Lys Ala Gln Gly Glu Trp Ser Glu Glu Leu Arg Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr Asp Ser Thr Ala Met Val Ser Trp Thr Ile Val Asp Gly Tyr Ser Ile Ser Ser Ile Ile Ile Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His Ile Asp Val Lys Ile Lys Asn Ala Thr Val Thr Gln Tyr Gln Leu Lys Gly Leu Glu Pro Glu Thr Thr Tyr His Val Asp Ile Phe Ala Glu Asn Asn Ile Gly Ser Ser Asn Pro Ala Phe Ser His Glu Leu Arg Thr Leu Pro His Ser Pro Gly Ser Ala Asp Leu Gly Gly Gly Lys Met Leu Leu Ile Ala Ile Leu Gly Ser Ala Gly Met Thr Cys Ile Thr Val Leu Leu Ala Phe Leu Ile Net Leu Gln Leu Lys Arg Ala A n Val Gln Arg Arg Met Ala Gln Ala Phe Gln Asn Arg Glu Glu Pro Ala Val Gln Phe Asn Ser Gly Thr Leu Ala Leu Asn Arg Lys Ala Lys Asn Asn Pro Asp Pro Thr Ile Tyr Pro Val Leu Asp Trp Asn Asp Ile Lys Phe Gln Asp Val Ile Gly Glu Gly Asn Phe Gly Gln Val Leu Lys Ala Arg Ile Lys Lys Asp Gly Leu Arg Net Asp Ala Ala Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly His His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly Tyr Leu WO 94/04694 PCr/CA93/00352 f 214333~ - 46 -yr Leu Ala Ile Glu q~yr Ala Pro His Gly Asn Leu Leu Asp Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Ile Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Leu Leu His Phe Ala Ala 930 g35 940 Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gln Lys Gln Phe Ile Hi~

rg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr Ile Ala ys I le Ala Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu Ser Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Thr Pro Tyr ~ys Gly Met hr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Leu Glu ys Pro Leu Asn Cys Asp Asp Glu Val Tyr Asp Leu Met Arg Gln Cys Trp Arg Glu Ly Pro Tyr Glu Arg Pro Ser Phe Ala Gln Ile Leu Val Ser Leu Asn Arg Met Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly Ile Asp Cys Ser Ala Glu Glu Ala Ala (2) INFORNATION FOR SEQ ID NO: 3:
( i ) SES; u~sN-:~ CT~RACTERISTICS:
( A ) LENGTH: 1590 base pairs (B) TYPEs nucleic acid (C) S~R~Nn~nN~SS: single ( D ) TOPOLOGY: 1 i n~:~r ( ii ) MoT~cuT~F! TYPE: cDNA

~ 94/~694 2 I ~ 3 3 3 ~ PCT/CA93/00352 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (D) DEVELOPMENTAL STAGE: Embryo (vii) IMMEDIATE SOURCEs (A) LIBRARY: murine embryonic lambda gtlO cDNA library (B) CLONE: 1.6kb clone (viii) POSITION IN GENOME:
(A) CHROMOSOME /.~GM~NT: 4 (B) MAP POSITION: Between the brown and pmv-23 loci (ix) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION: 1..903 ( Xi ) SEQ~N~ DESCRIPTION: SEQ ID NO:3:
ATC AAG TTT CAA GAC GTG ATC GGA GAG GGC AAC TTT GGC CAG GTT CTG

Ile Lys Phe Gln Asp Val Ile Gly Glu Gly Asn Phe Gly Gln Val Leu AAG GCA CGC ATC AAG AAG GAT GGG TTA CGG ATG GAT GCC GCC ATC AAG
96ys Ala Arg Ile Lys Lys Asp Gly Leu Arg Net Asp Ala Ala Ile Lys AGG ATG AAA GAG TAT GCC TCC AAA GAT GAT CAC AGG GAC TTC GCA GGA

Arg Met Ly3s5 Glu Tyr Ala Ser Lys Asp Asp His Arg Asp Phe Ala Gly GAA CTG GAG GTT CTT TGT AAA CTT GGA CAC CAT CCA AAC ATC ATT AAT

Glu Leu Glu Val Leu Cys Lys Leu Gly His His Pro Asn Ile Ile Asn CTC TTG GGA GCA TGT GAA CAC CGA GGC TAT TTG TAC CTA GCT ATT GAG

Leu Leu Gly Ala Cys Glu His Arg Gly Tyr Leu Tyr Leu Ala Ile Glu TAT GCC CCG CAT GGA AAC CTC CTG GAC TTC CTG CGT AAG AGC AGA GTG
288yr Ala Pro His Gly Asn Leu Leu Asp Phe Leu Arg Lys Ser Arg Val CTA GAG ACA GAC CCT GCT TTT GCC ATC GCC AAC AGT ACA GCT TCC ACA

Leu Glu Thr Asp Pro Ala Phe Ala Ile Ala Asn Ser Thr Ala Ser Thr WO94/~K94 PCT/CA93/00352 2l43,336 CTG TCC TCC CAA CAG CTT CTT CAT TTT GCT GCA GAT GTG GCC CGG GGG

Leu Ser Ser Gln Gln Leu Leu His Phe Ala Ala Asp Val Ala Arg Gly ATG GAC TAC TTG AGC CAG AAA CAG TTT ATC CAC AGG GAC CTG GCT GCC
432 :~
Met Asp Tyr Leu Ser Gln Lys Gln Phe Ile His Arg Asp Leu Ala Ala AGA AAC ATT TTA GTT GGT GAA AAC TAC ATA GCC AAA ATA GCA GAT TTT

Arg Asn Ile Leu Val Gly Glu Asn Tyr Ile Ala Lys Ile Ala Asp Phe GGA TTG TCA CGA GGT CAA GAA GTG TAT GTG AAA AAG ACA ATG GGA AGG
528ly Leu Ser Arg Gly Gln Glu Val Tyr Val Lys Lys Thr Net Gly Arg CTC CCA GTG CGT TGG ATG GCA ATC GAA TCA CTG AAC TAT AGT GTC TAT
576eu Pro Val Arg Trp Net Ala Ile Glu Ser Leu Asn Tyr Ser Val Tyr ACA ACC AAC AGT GAT GTC TGG TCC TAT GGT GTA TTG CTC TGG GAG ATT

Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly Val Leu Leu Trp Glu Ile GTT AGC TTA GGA GGC ACC CCC TAC TGC GGC ATG ACG TGC GCG GAG CTC

Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly Met Thr Cys Ala Glu Leu TAT GAG AAG CTA CCC CAG GGC TAC AGG CTG GAG AAG CCC CTG AAC TGT

Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Leu Glu Lys Pro Leu Asn Cys GAT GAT GAG GTG TAT.GAT CTA ATG AGA CAG TGC TGG AGG GAG AAG CCT
768sp Asp Glu Val Tyr Asp Leu Met Arg Gln Cys Trp Arg Glu Lys Pro TAT GAG AGA CCA TCA TTT GCC CAG ATA TTG GTG TCC TTA AAC AGG ATG
816yr Glu Arg Pro Ser Phe Ala Gln Ile Leu Val Ser Leu Asn Arg Net CTG GAA GAA CGG AAG ACA TAC GTG AAC ACC ACA CTG TAT GAG AAG TTT

Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr Thr Leu Tyr Glu Lys Phe ~ 94/0~94 PCT/CA93/00352 2I~3336 ACC TAT GCA GGA ATT GAC TGC TCT GCG GAA GAA GCA GCC TAGAGCAGAA

Thr Tyr Ala Gly Ile Asp Cys Ser Ala Glu Glu Ala Als CATGT ACAA~r7GCCA lll~l~CTCA CTGGCGCGAG AGCCTTGACA CcTG~AccAA

GCAAGCCACC CACTGCCAAG AGATGTGATA ~A~AAGTGTA TATATTGTGC l~ GGG

ACCCTCCTCA TACAGCTCGT GCGGATCTGC AGl~lGll~l GACTCTAATG TGACTGTATA

TACTGCTCGG AGTAAGAATG TGCTAAGATC AGAATGCCTG TTCGTGGTTT rATATAATAT

AlllllLlAA AAGr~TAr~AT TGcArAGr~AA GGTATGAGTA CA~ATAcTGT AATGrA~AAc GllATTGT CCTAGATGTG TTTGACATTT TTCCTTTACA ACTGAATGCT A~AAAAGTGT

TTTGL-l~-l-G~l~GCGCGTAAGA TAC~G-~'C~-. ~AAAA~AAGC ATTCCCTTGA rAr7rArAG&A

AGAAAAGCGA ÇÇr,AAATGTA TGGATTATAT TAAA~ TTAc~AcArA AGAGG~CGAA

CATTCCAAGT AGCAr-AAr-AG AGG~l~l~lC AACTCTGCTC CTCACCTGCA GAAGCCAGTT

GGCCA TGTr-ACAATT GTCC~Gl~ll TTTAT~GrAr, Cr.-AAATCATT C~AAAA~ATG

AACATCTAAA AACTTTGCTA Gr-AGACTAAG AAccTTTGGA ÇAr-A~AGA~A TAAGTACG&T

rAAAAAArAA AACTGCG

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQ~N~ r. ~ARACTERISTICS:
'A' LENGTH: 301 amino acids B TYPE: amino acid ,D; TOPOLOGY: lineAr (ii) MOLECULE TYPE: protein (xi) SEQu ~:NL~ DESCRIPTION: SEQ ID NO:4:
Ile Lys Phe Gln Asp Val Ile Gly Glu Gly Asn Phe Gly Gln Val Leu WO 94/04694 PCr/CA93/00352 2l4333~

Lys Ala Arg Ile Lys Lys Asp Gly Leu Arg Met Asp Ala Ala Ile Lys Arg Net Lys Glu Tyr Ala Ser Lys Asp Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly His His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly Tyr Leu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly Asn Leu Leu Asp Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Ile Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Leu Leu His Phe Ala Ala Asp Val Ala Arg Gly ~et Asp Tyr Leu Ser Gln Lys Gln Phe Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr Ile Ala Lys Ile~la Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu Ser Leu Asn Tyr Ser Val Tyr = 180 185 190 Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Thr Pro ~ryr Cys Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gln Gly Tyr Arg Leu Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr Asp Leu Met Arg Gln Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro Ser Phe Ala Gln Ile Leu Val Ser Leu Asn Arg Met Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly Ile Asp Cys Ser Ala Glu Glu Ala Ala 94/~94 2 1 ~ 3 3 3 $ PCT/CA93/00352 (2) INFORMATION FOR SEQ ID NO:5:
~ (i) SEQUE~CE CHARACTERISTICS:
(A) LENGTH: 847 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: Nus musculu~
(vii) IMMEDIATE SOURCE:
(A) T.TR~A~y mouse genomic bacteriophage library (viii) POSITION IN GENOME:
(A) cHRoMosoME/~r/M~T: 4 (B) MAP POSITION: Between the brown ~nd pmv-23 loci (xi) SEQu~N~ DESCRIPTION: SEQ ID NO:5:
GCAAGTGCTG CTCCCCGTGC ccrAAAr-rcc C~ CAG GGATrCC~AA Tr-rACrCCAG

ArAAcAGcTT AGCCTGCAAG GGCl~lCCT cATcr7r~Ac C~TACATAGT GGAGGCTTGT

TATTCAATTC CTGGCCTATG A~AGr-ATACC CCTA~ C CTr-AAAATGC TGACCAGGAC

CTTA~~ A ACAAAr~ATCC CTCTGCrCCA CAATCCAGTT AAGGC~Gr-AG cAGr7A-ccG5A

GCAGGAGCAG AAGA~AAr,CC TTGGATGAAG GGrAAGRTGG ATAGGGCTCG CTCTGCCCCA

AGCCCTGCTG ATACCA~r~TG CCTT~AA~A~ ACAGCCTTTC CCATCCTAAT cTrJcAAArGA

AACAGGAAAA AGGAACTTAA CCCTCCCTGT GCT~AGACAG AAATGAGACT GTTACCGCCT

GCl~ GG ~ lClCCT TGCCGCCAAC TTG~AAA~AA GAGCGAGTGG ACCATGAGAG

CGGGAAGTCG CAAA~l.~lG A~ll~llGAA AGCTTCCCAG GGACTCATGC TCAl~l~l~G

ACGCTGGATG r~GAGATCTG r-Gr-AAGTATG GAC-l~-l-l-lAG CCGGCTTAGT~ GA

GTCAGCTTGC TCCTTTATGG TAAG~ G CTTGATGTTT All~l~l~lGl GlGlGlCATG

W094/~g4 PCT/CA93/00352 ~

2~ ~333 ~ 52 -TTT~AACAAC AGTGACTTCT CGCCATTCTC l~l~lCACCA AACCTTCGAT TTGGTGACCC

TGACACTGCT lll~lC,AGAC TCTCCAGTTT ACACATGGCA ACGGllll~A AGTTCAGATT

CcAGc~GrAc CAGCTGGTTT TrA~,~CATCT'l~ ,lAGAC AGATGCTGCC TTCCl~

GCCACGG

Claims (26)

WE CLAIM:

1. An isolated transcriptional regulatory element which is capable of directing expression of a gene in cells of the endothelial lineage comprising a 7.2 kb fragment which extends from the Bgl II restriction site of the sequence to the Kpn I
restriction site as shown in Figure 11.

2. An isolated transcriptional regulatory element comprising (a) a nucleotide sequence having nucleotides 1 to 560 of the sequence as shown in the Sequence Listing as SEQ. ID. NO.5 and (b) nucleotide sequences complementary to (a).

3. A recombinant molecule adapted for transformation of a host cell comprising a transcriptional regulatory element as claimed in claim 1 and a gene operatively linked thereto.

4. A recombinant molecule adapted for transformation of a host cell comprising a transcriptional regulatory element as claimed in claim 2 and a gene operatively linked thereto.

5. A recombinant molecule as claimed in claim 3, wherein the gene is not a tek gene.

6. A recombinant molecule as claimed in claim 4, wherein the gene is not a tek gene.

7. A recombinant molecule as claimed in claim 3 wherein the gene is a reporter gene.

8. A recombinant molecule as claimed in claim 4 wherein the gene is a reporter gene.

9. A recombinant molecule as claimed in claim 7 wherein the reporter gene is a lac? gene which codes for .beta.-galactosidase, a neo gene which codes for neomycin phosphotransferase, a cat gene which codes for chloramphenicol acetyltransferase, a dhfr gene which codes for dihydrofolate reductase, a aphIV gene which codes for hygromycin phosphotransferase, a lux gene which codes for luciferase, or uidA gene which codes for .beta.-glucuronidase.

10. A recombinant molecule as claimed in claim 8 wherein the reporter gene is a LacZ gene which codes for .beta.-galactosidase, a neo gene which codes for neomycin phosphotransferase, a cat gene which codes for chloramphenicol acetyltransferase, a dhfr gene which codes for dihydrofolate reductase, a aphIV gene which codes for hygromycin phosphotransferase, a lux gene which codes for luciferase, or a uidA gene which codes for .beta.-glucuronidase.

11. A recombinant molecule as claimed in claim 3 wherein the gene encodes a toxic or therapeutic substance or an angiogenic factor.

12. A recombinant molecule as claimed in claim 4 wherein the gene encodes a toxic or therapeutic substance or an angiogenic factor.

13. A recombinant molecule adapted for transformation of a host cell comprising a transcriptional regulatory element as claimed in claim 1 operatively linked to a gene and a reporter gene.

14. A recombinant molecule as claimed in claim 13, wherein the gene is not a tek gene.

15. A transformant host cell including a recombinant molecule as claimed in any one of claims 3 to 14 with the proviso that the recombinant molecule does not contain a tek gene from the same species as the host cell.

16. A cell line comprising transformant host cells including a recombinant molecule as claimed in any one of claims 3 to 14 with the proviso that the recombinant molecule does not contain a tek gene from the same species as the host cell.

17. A method of producing a transgenic non-human mammal characterized as having a plurality of cells containing a recombinant molecule as cliamed in any one of claims 5, 6, 11, 12, and 19, or an ancestor of the mammal at an embryonic stage, comprising (a) introducing a recombinant molecule as claimed in any one of claims 5, 6, 11, 12 and 19 into a pronucleus of a mammlian zygote by microinjection, said zygote being capable of development into a mammal, thereby obtaining a genetically transformed zygote; (b) transplatating an embryo derived from the genetically transformed zygote into a pseudo-pregnant female capable of bearing the embryo to term and (c) if desired allowing the embryo to develop to term.

18. A method of determining the affect of a substance on cells of the endothelial lineage comprising producing a transgenic non-human mammal, an embryo thereof, or an ancestor of the mammal at an embryonic stage, characterized as having a plurality of cells containing a recombinant molecule comprising the transcriptional regulatory element as claimed in claim 1 operatively linked to a gene encoding the substance comprising (a) introducing the recombinant molecule into a pronucleus of a mammalian zygote by microijection, said zygote being capable of development into a mammal, thereby obtaining a genetically transformed zygote; (b) transplanting an embryo derived from the genetically transformed zygote into a pseudo-pregnant female capable of bearing the embryo to term and (c) isolating the enbryo or allowing the embryo to develop to term, and (d) determining the affect of the substance on cells of the endothelial lineage by comparison to a control.

19. A method of determining the affect of a substance on cells of the endothelial lineage as claimed in claim 18 wherein the recombinant molecule additionally comprises a reporter gene encoding a phenotype which is not displayed by the mammal, or an ancestor of the mammal at an embryonic stage and wherein after step (c) the phenotype of the reporter gene in the embryo or transgenic non-human mammal is assayed to determine the pattern and extent of expression of the gene.

20. A transgenic non-human mammal all of whose germ cells and somatic cells contain a recombinant molecule introduced into the mammal, or an ancestor of the mammal at an embryonic stage, the recombinant molecule comprising a transcriptional regulatory element as claimed in claim 1 or 2 operatively linked to a gene which is not the tek gene.

21. A method of using the transcriptional regulatory element as claimed in claim 1 in gene therapy to introduce a foreign gene into endothelial cells to correct or prevent vascular disorders.

22. . The method as claimed in claim 21 wherein the vascular disorders are hypertension, atherosclerosis, arthritis, restenosis, and cancer.

23 . The method as claimed in claim 21 or 22 wherein the foreign gene encodes a therapeutic agent preferably an anticoagulant, vasodilator or angiogenic factor.

24. A method of using the transcriptional regulatory element as claimed in claim 1 in gene therapy to introduce a foreign gene into endothelial cells to treat systemic or inherited disorders.

25. A method of using the transcriptional regulatory element as claimed in claim 1 or 2 to express a foreign gene in mammalian cells of the endothelial lineage.

26. A method as claimed in claim 25 wherein the transcriptional regulatory element is used to express a foreign gene in an embryonic or adult mouse.