| First Author | Paris D | Year | 1995 |
| Journal | Mouse Genome | Volume | 93 |
| Issue | 4 | Pages | 1038-40 |
| Mgi Jnum | J:30569 | Mgi Id | MGI:78686 |
| Citation | Paris D, et al. (1995) A comparison between and inbred strain and hybrid lines to generate transgenic mice. Mouse Genome 93(4):1038-40 |
| abstractText | Full text of Mouse Genome contribution: A COMPARISON BETWEEN AN INBRED STRAIN AND HYBRID LINES TO GENERATE TRANSGENIC MICE. D. PARIS, K. TOYAMA, P.M. SINET, P. KAMOUN and J. LONDON. URA CNRS 1335. Universite Rene Descartes, Hopital Necker, 75743 Paris Cedex 15 France. Tel: 33 144 49 47 54, Fax: 33 142 730 659. Introduction Several different hybrids are currently approved for DNA microinjection, and the C57BL/6 x SJL Fl hybrid has been shown to be efficient in the production of transgenic mice (1). The majority of the hybrids used for microinjection utilize the C57BL/6 inbred strain as one of the parental stocks because of favourable genetic and embryological characteristics. Hybrid mice are often used because hybrid vigour not only imparts desirable reproductive characteristics but also enhances the egg quality, hence leading to desirable microinjection characteristics. However, one might be concerned with the uniformity of the genetic background in which the transgene will be functioning. The initial selection of outbred strains for gene transfer work focused on the efficiency of maintenance and reproduction as well as known embryology and response to experimental manipulations. However, current applications necessitate transgenic mouse models produced in specific background strains. Significant variation in transgene expression between individuals, litters, or generations can doom a transgenic model at several stages, and can complicate the initial characterisation of the model if the genetic background severely influences the transgene expression (2, 3). Moreover, it has been observed (4) that particular homozygous transgenic knock-out mice died in some genetic backgrounds and were viable in other ones. Alternatively, as the model is bred through several generations, inbreeding can alter transgene expression slowly or bring out new recessive phenotypes unrelated to the transgene. In transgenic experiments, random integration of the transgene can induce new mutation (5). If Fl or F2 outbred zygotes are used, analysis will be complicated by the hybrid nature of the mutant animals, and extensive backcrossing will be needed to establish mutants with an inbred background. We compared for two different transgenes having similar size (around 12 kb) the reproductive performance, the survival of embryos after DNA injection and the efficiency of generating transgenic mice with FVB/N inbred strain, B6SJL and B6CBA hybrid strains. Material and methods Mice: A pair of FVB/N mice (originated from the Jackson Laboratory) were kindly provided in 1991 by Dr J.L. Guenet (Pasteur Institute, France) and were used to produce FVB/N mice in our animal house. SJL and C57BL/6 mice (4-5 weeks old) were obtained from CSAL CNRS Orleans (France) and were cross- breeding at 2-3 months in our animal colony to produce C57BL6/SJL (B6SJL) mice. C57BL6/CBA (B6CBA) mice (4-5 weeks old) were obtained from Janvier (France) and NMRI mice (4-5 weeks) were originated from Iffa Credo (France). Generation and collection of embryos: FVB/N (5-6 weeks old), B6SJL Fl and B6CBA Fl female mice (4-5 weeks old) were superovulated by I.P injection of 5 I.U of Pregnant Mares Serum Gonadotrophin (PMSG; Centravet Laboratories) at 11.30 am and 5 1.U Human Chorionic Gonadotrophin (HCG; Intervet Laboratories) 48 hours apart. Animals were maintained in a 12 hours dark cycle 7.00 pm-7.00 am. To obtain embryos, FVB/N, B6SJL Fl and B6CBA Fl females were paired individually overnight with FVB/N males, B6SJL Fl and B6CBA Fl males respectively (2-10 months old). The females were then inspected for vaginal plugs the next morning as an indication of successful mating. Oviducts were dissected on the day of plugging into E.T medium (Gibco, BRL) prewarmed to 37 degrees C. The cumulus masses released were then treated for a few minutes in E.T medium supplemented with 0.1 M hyaluronidase (Sigma). The 1-cell zygotes were recovered, washed in E.T medium and cultured in INRA B2 Menezo medium (Biomerieux, France) at 37 degrees C in 5% CO2 until the microinjection step. Production of transgenic mice: Production of transgenic mice is ensured by the classical pronuclear microinjection procedure (6). Two different transgenes of similar size were used. First a linear 11.5 kb EcoRI-BamHI human DNA fragment encompassing the entire human Cu/Zn superoxide dismutase gene (hSOD-1 construct) (7) and second, a linear 13.5 kb EcoRV DNA fragment of the Beta-CEP4-delta-NL plasmid (8) containing the human cDNA coding for the amyloid precursor protein (APP) with the Swedish mutation specific for familial Alzheimer disease, linked to the strong cytomegalovirus promoter and to a simian virus 40 polyadenylation sequence (APP construct). DNA solutions were injected at a concentration of 2 ug/ml in 8 mM pH=7.5 Tris-HC1/0.1 mM EDTA, so that, approximately 200 to 400 copies of transgenes were injected into the male pronucleus of zygotes. Embryos that survived microinjection were reimplanted the same day into pseudopregnant NMRI females (2-3 months old) previously random mated to NMRI vasectomized males. Statistical analysis: comparisons between means and between percentage were performed by t test and by x2 test respectively. Results Reproductive performance of FVB/N, B6SJL and B6CBA strains: Two outbred strains (B6SJL, B6CBA) and one inbred strain of mice (FVB/N) were used to produce embryos (as described in Material and Methods). As shown in Table 1, the number of eggs recolted per female after superovulation is lower for FVB/N compared to B6SJL (P<10-5) and B6CBA female mice (P<10-9), 56.3%, 63.5% and 71.4% of the embryos from FVB/N, B6SJL and B6CBA mice respectively were fertilized and were subjected to DNA injection. FVB/N mice produced less microinjectable embryos than B6SJL mice (P<l0-4), and than B6CBA mice (P<10-9). Table I: Reproductive performance of FVB/N, B6SJL Fl and B6CBA Fl mice. Zygote strain: FVB/N; Isolated oocytes per female: 18.0 +/- 5.1; total number: 5284; Number of injectable oocytes (% of total): 2997 (56.3%). Zygote strain: B6SJL F2; Isolated oocytes per female: 19.4 +/- 3.4; total number: 1994; Number of injectable oocytes (% of total): 1266 (63.5%). Zygote strain: B6CBA F2; Isolated oocytes per female: 25.2 +/- 11.5; total number: 1484; Number of injectable oocytes (% of total): 1059 (71.4%). Survival of embryos after DNA injection: As shown in table II, the survival of embryos after microinjection is lower for FVB/N embryos than for B6SJL (P<l0-3) or B6CBA embryos (P<10-3). It has to be pointed out that the microinjection procedure is facilitated by the fact that inbred FVB/N embryos have more prominent pronuclei and clearer cytoplasm compared to the two outbred lines used (9). In the other hand, the plasma membrane of FVB/N embryos shows more flexibility during the introduction of microinjection pipette compared to outbred embryos, and this phenomenon could explain the more important vulnerability of FVB/N eggs after microinjection. Table II: Efficiency of transgenesis using FVB/N, B6SJL F2 and B6CBA F2 zygotes. Zygote strain: FVB/N; DNA construct: hSOD-1; Number survived eggs (% of injected eggs): 391 (58.0%); Transfered eggs per female: 14.7 +/- 1.6; *total number: 324; Number of implanted embryos (% of transferred eggs): 77 (23.8%); In-utero resorbed embryo (% of implanted embryos): 19 (24.7%); Newborns per litter: 4.8; total number: 58; Transgenic mice number (% of newborns): 1 (1.7%). Zygote strain: B6SJL F2; DNA construct: hSOD-1; Number survived eggs (% of injected eggs): 844 (66.7%); Transfered eggs per female: 15.2 +/- 2.6; *total number: 668; Number of implanted embryos (% of transferred eggs): 151 (22.6%); In-utero resorbed embryo (% of implanted embryos): 41 (27.2%); Newborns per litter: 5.7; total number: 110; Transgenic mice number (% of newborns): 2 (1.8%). Zygote strain: FVB/N; DNA construct: APP; Number survived eggs (% of injected eggs): 985 (42.8%); Transfered eggs per female: 18.1 +/- 1.8; *total number: 543; Number of implanted embryos (% of transferred eggs): 119 (21.9%); In-utero resorbed embryo (% of implanted embryos): 17 (14.3%); Newborns per litter: 4.3; total number: 102; Transgenic mice number (% of newborns): 2 (2.0%). Zygote strain: B6CBA F2; DNA construct: APP; Number survived eggs (% of injected eggs): 523 (49.4%); Transfered eggs per female: 17.0 +/- 1.8; *total number: 543; Number of implanted embryos (% of transferred eggs): 67 (19.8%); In-utero resorbed embryo (% of implanted embryos): 12 (17.9%); Newborns per litter: 5.5; total number: 55; Transgenic mice number (% of newborns): 1 (1.8%). *Only a part of injected eggs was transfered depending on the pseudopregnant mice available. Long term survival of microinjected embryos: A similar number of FVB/N, B6CBA and B6SJL zygotes were transferred after micro- injection and 1 to 2 hours in culture, the same day onto NMRI pseudo- pregnant mice (Table II) known to be particularly good foster mothers. The number of implanted embryos was determined after the birth of pups by uterus examination of NMRI recipient females, and the number of in-utero resorbed embryo deduced by subtracting the number of implanted embryos from the number of new-borns obtained (Table II). Overall, there is no significant difference between the number of implanted embryos from FVB/N and the outbred strains. No strain difference effect was observed for the number of in utero resorbed embryos. As shown in Table II, the number of transgenic mice obtained for the different strains used is identical. Discussion FVB/N strain was used in this study because it presents a superior fecundity compared with most inbred strains of mice (9), but a reduced number of injectable FVB/N zygotes was obtained compared to that of the two outbred strains. However, FVB/N embryos harboured prominent pronuclei and more translucent cytoplasm than the hybrids used, and then facilitated the micro- injection step. In contrast to Taketo et a1 (9) findings, we observed that the survival of FVB/N embryos was reduced after injection of DNA compared to outbred zygotes. Nevertheless, efficiency of generating transgenic mice is similar for the FVB/N inbred and the two hybrid strains used, according to Taketo et a1 results (9). Low frequency (~2%) of transgenic offsprings was obtained depending on the nature of the transgene used. As a matter of fact, it is known some transgenes could have deleterious properties leading to a reduced efficacity in generating transgenic mice (10). The use of outbred strains is widely documented, but it might be a handicap for some studies (especially behavioural and neuro- anatomical where the genetic background is significantly implicated), since all the transgenic mice obtained from a founder transgenic animal have different genetic backgrounds. Furthermore, the control mice originated either from the hybrid strain used or from non transgenic animals of a same litter were obviously different genetic backgrounds. Although efficiencies in inbred mice (related to superovulation, microinjection, and reproduction) are habitually reduced, the utility associated with genetic characterization adds a dimension that may be required for particular projects. Thus, in spite of some drawbacks, the use of FVB/N inbred strain to generate transgenic mice presents several advantages. Acknowledgements We wish to thank Dr S. Younkin for the gift of the APP construct. D. Paris is supported by grants from France Alzheimer Association and from the Chancellerie des Universites de Paris. This work was supported by Centre National de la Recherche Scientifique, AFM, Ministere de la Recherche et de l'Enseignement Superieur, Universite Paris V and Faculte Necker. References I. Brinster, R.L. et al. Proc. Natl. Acad. Sci. U.S.A., Vol. 82, pp. 4438-4442, 1985. 2. Chisari, F.V. et al. Cell, Vol. 59, pp. 1145-1156, 1989. 3. Harris, A.W. et al. J. Exp. Med., Vol. 167, pp. 353-371, 1988. 4. Baribault, H et al, Genes & Development, Vol. 8, pp. 2964-2973, 1994. 5. Meisler, M.H et al. Trends in Genetics, Vol.8, pp. 341-344, 1992. 6. Hogan, B. et al. Cold Spring Harbor 1986. 7. Hallewell, R. et al. In ÔSuperoxide dismutase in chemistry, biology and medicine', Rotilio, G, ed., pp. 249-256, 1986. 8. Cai, X.D. et al. Science, Vo1. 259, pp. 514-516, 1993. 9. Taketo, M. et al. Proc. Natl. Acad. Sci. USA, Vo1. 88, pp. 2065-2069, 1991. 10. Simson, J. et al. Lab Invest, Vo1. 71, pp. 680-687, 1994. |
