1. Abstract: Number of copies, germ line transmission, and expression level of a GFP transgene in mice generated by lentiviral vectors Teixeira M. *1, Nègre D. *2,3, Blanquier B. 4, Ghittoni R. 5, Szecsi J. 3, Henry F. 1, Coupet CA. 5, Aubert D. 1, Cosset FL 3, Marvel J. 5 1.PBES,Ecole Normale Supérieure de Lyon, 2.Plateau de vectorologie - Ecole Normale Supérieure de Lyon, 3.Unité U758 Inserm – ENS Lyon, 4.Plateau de QPCR – CERVI- Lyon, 5.Unité U851, INSERM Lyon * Both authors contributed equally to this work Abstract: Up to now transgenic mice were mainly obtained by pronuclear microinjection of DNA. However, recently an alternative method based on the use of lentiviral vector has been developped, providing better efficiency of trangenesis. The aim of this work was to genetically characterize the founders and their progeny obtained by this new method. Also, we tried to correlate the expression level obtained using three ubiquitous promoters - CMV early enhancer/chicken β actin hybrid promoter (CAG), spleen focus-forming virus promoter (SFFV) and ubiquitin C promoter (FG12) - in blood circulating lymphocytes, with the number of integrated copies (Fig 4). Lentiviral vectors carrying a GFP transgene were injected into the perivitelline space of fertilized oocytes, resulting in 52% to 80% of founder animals carrying the transgene, according to the titre of lentivirus vector preparations. The transmission of transgene to offspring by founders was also analysed. We used quantitative PCR to determine the number of copies of integrated transgene. The number of copies varies between 0.1 and 50, and is directly correlated with the concentration of the lentiviral vectors (Table 1). We found that the founders harbouring less than one copy are mosaics, indeed, all their transgene positive progeny harboured 1 copy (Fig 2). These results indicate that viral integration can occur at 2 and 4 cells stages. Mosaïcisme was not restricted to low viral titre as some F1 mice present a higher number of integrated copies than their founder. These results suggest that most founders obtained by this lentiviral transduction method, even those with many integrated copies, could be mosaics. GFP expression stability over generation was analyzed by flow cytometry, in peripheral blood lymphocytes of founders or transgenic animals. Expression of GFP seems to be stable during generations. Nevertheless, we noticed, that depending on the insertion site, in certain animals the expression could fade out with time (Fig 3 and fig 4). In conclusion, lentiviral transduction allows to quickly obtain a large number of transgenic mice, widely different genetically in regard of the transgene integration. However, mosaicisme seems to be a common feature of this approach and should be taken into account when analysing founder mice. Materials and Methods 1.Generation of transgenic mice by perivitelline injection of fertilized oocytes. 2.Extraction of DNA 3.Determination of copies number by quantitative PCR 4.Blood samples collection 5.Extraction of lymphocytes 6.Determination of GFP fluorescence in lymphocytes RESULTS 0 Copy number / cell 0,5 1 Example 1Example 2Example 3Example 4 A Fonder (F0)‏F1 Fig. 2 : The Founders are mosaics Transgene copy number of 5 founders and their newborns. (0.9 copy is considered as 1 copy) ‏ A: Founders with less than 1 copy of transgene per genome give rise to offspring with 1 GFP copy / genome B: When the founder have multiple copy, we also observe an increase of copy number in most of F1 mice 8,4 11,2 10,1 10,7 8,9 11,9 9,2 1,4 1,6 2,3 9,2 10,7 0 2 4 6 8 10 12 14 Copy number / cell B Example 5 Fig. 3: GFP expression in F0, F1 and F2 mice Analysis of GFP expression in blood lymphocytes in three generations (F0, F1, F2) of mice harbouring one GFP copy. Backcross of 25F0 mouse with wild type resulted in 6 transgenic mice with 1 transgene copy: 78F1, 41F1, 36F1, 82F1 and 77F1. All F0 and F1 animals and most of F2 mice express GFP in blood lymphocytes. 0 10 20 30 40 50 60 70 80 90 100 25F0 78F1 41F1 36F1 35F182F1 77F1 % of GFP positive cells Fonder (F0)‏F1 F2 Copy number/cell 0102030405060 % of grenn lymphocytes 100 0 10 20 30 40 50 60 70 80 90 SFFV FG12 CAG (C57Bl6)‏ CAG Fig. 4: GFP expression in Founders’ lymphocytes Correlation between GFP expression level in the lymphocytes and transgene copy number in the founders. CAG is the strongest promoter. 50-85% < 5 kb not limited multiple sites yes 10% not limited limitided 1 site yes % transgenic mice transgene size embryo background insertion site Germline transmission Lentiviral vectorsDNA injection Comparison of the two technics vector Viral titer (UI/ml)‏ Implanted embryos Newborn animals Transgeni c animals Efficien cy GFP expessing animals Copy number FG12-gfp 6.4 10 8 93231774%110.4-50 SFFV-gfp 1.8 10 9 102241980%170.2/37 CAG-GFP 106251352%110.14-1.5 SFFV- OVONP6 8-IRES- 4.9 10 8 524301961%00.2-10 8 10 7 34014964%90.2-1.4 Table 1: Summary of results obtained with different vectors Embryo viability and rates of implantation, transgenesis and expression. FG12: Ubiquitin C. SFFV: Spleen Focus-Forming Virus. CAG: CMV early enhancer/chicken β actin hybrid promoter CAG-GFP ( C57Bl6)‏ 8 10 7