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In order to determine whether the cloned 14 Kb Ly-6E. 1 gene was capable of functioning in vivo, the jS-galactosidase reporter gene was inserted into the first, untranslated exon of Ly-6E. 1 to enable detection of expression from the transgene. The cloning strategies employed are represented in figure 1. To facilitate the introduction of reporter genes into the complete 14 Kb cloned Ly-6E .l genomic fragment, pL6Cla was generated by linking pClal, consisting of the 5’ 3.5 Kb fragment of the Ly-6E .l gene with a unique Clal site engineered into the first exon (Sinclair, Daly & Dzierzak, 1996), to a 12.3 Kb EcoRl(partial) - Sphl fragment from pAB14 containing the remaining 3’ part of the Ly-6E. 1 gene in pPolylll. The Smal restriction site 3’ of the lacZ gene in p610ZA (gift D. Meijer) was converted into a Narl site using synthetic oligonucleotides, enabling the isolation of lacZ coding and SV40 polyadenylation sequences as a 3.6 Kb Narl fragment which was then cloned into the unique Clal site in pL6Cla to generate pL6LZ. The orientation of the insert was checked by restriction digest using EcoRV, an enzyme whose site is as^ymetrically located within the lacZ gene.

Figure 1. Cloning strategy used to generate the Ly-6E. HlacZ construct.

Schematic representation of the cloning steps involved in linking pClal and pAB14 to generate pL6Cla and subsequent insertion of a Narl flanked lacZ gene into the unique Clal cloning site within the first exon of Ly-6E .l.

pClal = 3.5 Kb BamHl-EcoRl Ly-6E .l fragment with unique Clal restriction site within first exon (Sinclair et al. y 1995); pAB14 = original 14 Kb BamHl subclone of Ly-6E .l in pPolylH (Sinclair & Dzierzak, 1993); p610ZA = lacZ gene with SV40 polyadenylation signal downstream of hsp67 promoter in pUC13 (gift. D. Meijer); pL6LZ = Ly-6E.l//ûcZ construct in pPolylll, from which a 17.6 Kb Notl fragment was used for microinjection to generate transgenic mice.

Restriction enzyme sites: R1 = EcoRl; C = Clal; S = Sphl; N = Notl; B = BamHl; Sm = Smal; Na = Narl; RV = EcoRV. PA = SV40 polyadenylation signal.

For generation of Ly-6E. Hmyc and Ly-6E. l/bcl-2 constructs, the c-myc and bcl-2 genes respectively were flanked with Clal compatible restriction sites and cloned into pL6Cla as with lacZ.

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To verify that the resulting Ly-6E. \HacZ transgene (pL6LZ) was capable of producing functional lacZ protein, it was transfected into an Ly-6E .l^ cell line, NIH3T3. pPolyin vector sequences were removed entirely by digestion with Notl and the resulting 17.6 Kb fragment (figure 2A) was purified by preparative gel electrophoresis prior to being cotransfected into NIH3T3 cells with a neomycin resistance expression vector. G418 resistant clones transfected with control pAB14 (figure 2B) or with pL6LZ (figure 2C) were stained with X-gal. Only transfectants containing pL6LZ show expression of functional jS-galactosidase.

For the production of transgenic mice, the 17.6 Kb Notl Ly-6E.l//ocZ fragment (figure 2A) was microinjected into pronuclei of fertilised (CBA/Ca x C57/B10) F2 oocytes. 30 oocytes not visibly damaged by microinjection were transferred into one oviduct of each pseudopregnant female recipient. In general, one of three pseudopregnant recipients produced offspring, of which approximately one of four carried the transgene. Litter size of founder mice ranged between 1 and 5. Genomic DNA prepared from tail biopsies obtained from founder animals 10 days post partum was screened by southern blot for the presence of the lacZ transgene. Of a total of 34 founder mice bom after microinjection of Ly-6E. HlacZy five were positive for the lacZ transgene. From these five transgenic founders, a total of four Ly-6E.l//dcZ lines of transgenic mice were generated. Figure 2D shows a southern blot, used to determine transgene copy number, of BamHl restricted genomic DNA from transgenic animals of Ly-6E. HlacZ lines probed with a lacZ probe and a Thy-1 probe. BamHl digestion of Ly-6E. lUacZ yields a 3.6Kb lacZ fragment, the intensity of which is normalised for loading using the hybridisation signal of a 14Kb fragment of the endogenous Thy-1 gene. After adjusting for loading differences, copy number

Figure 2. LacZ expression from the Ly-6E. \HacZ construct and the generation of transgenic mice.

A. Diagram of the Ly-6E. l//ocZ construct compared with the Ly-6E. 1 gene showing the location, in Kb, of DNase 1 hypersensitive sites relative to transcriptional start site (after Sinclair & Dzierzak, 1993). B = BamHl and X = Xbal restriction sites.

B&C. X-gal stained G418 resistant colonies of NIH3T3 cells after transfection with SV40neo alone (B.) or Ly-6E .l//ûcZ plus SV40neo (C.).

D. Southern blot of 5fig BamHl digested DNA from lines BLla, BLlb, BLRl, BL7 and BL19 run on a 1 % agarose/TAE gel. BamHl digestion removes the lacZ gene and SV40 polyadenylation signal from Ly-6E.l//ocZ as a 3.3 Kb fragment and cleaves the endogenous Thy-1 gene, used as a loading control in this case, to yield a 14 Kb fragment. After blotting, the filter was hybridised with ^^-labelled lacZ (1.1 Kb EcoRV-BamHl) and Thy-1 (1.2 Kb Xbal-N rul) probes. Known amounts of pL6LZ in 5/tg of non-transgenic DNA was used for copy number controls. The numbers to the right of the blot correspond to the distance migrated by BstEn digested X DNA size marker.

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D Thy 1 - :9 E 2 _ i _ j _ j _ j _ j OÛ Dû C û CÛ CÛ Controls Lac Z - Copy # 4 4 5 6 1 5 0 1 5 1 0 20 14 7.2 6.4 4.8 4.3 3.7 2.3

was determined based upon comparison with controls generated from dilutions of pL6LZ representative of specific numbers of transgene copies per genome (Ipg plasmid gives an equivalent hybridisation signal to 1 transgene copy in Ifig genomic DNA). Quantitation of hybridising signals was carried out using a Phosphorimager (Molecular Dynamics). From such calculations, the number of copies per genome were estimated as follows: BLla, 4; BLlb, 4; BL7, 6 and BL19, 15. In addition, a single transgenic founder, BLRl containing 5 copies of the transgene was included in the analysis but this animal was unable to establish a transgenic line and was considered to be sterile. Copy number analysis of genomic DNA from all BLRl tissues analysed was performed to determine the level of chimaerism in each. A similar level of transgene contribution (5 copies) was found in each tissue, confirming that BLRl was not mosaic, ie, not chimaeric for transgenic/non-transgenic cells in any tissue (data not shown).