Abstract
Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.
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References
Brem, G., Wanke, R., Wolf, E., Buchmuller, T., Brenig, B. and Hermanns, W. (1989) Multiple consequences of human growth hormone expression in transgenic mice.Mol. Biol. Med. 6, 531–47.
Brem, G., Springman, K., Meier, E., Kraublich, H., Brenig, B., Muller, M. and Winnacker, E.L. (1990). Factors in the success of transgenic pig programs. InTransgenic Models in Animal Agriculture pp. 61–72. Boston, MA: Wiley Liss.
Brinster, R.L., Richie, K.A., Hammer, R.E., O'Brien, R.L., Arp, B. and Storb, U. (1993) Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice.Nature 306, 332–6.
Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K. and Palmiter, R.D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice bymicroinjecting eggs.Proc. Natl Acad. Sci. USA 82, 4438–42.
Burki, K. and Ulrich, A. (1982) Transplantation of the human insulin gene into fertilized mouse eggs.EMBO J. 1, 127–31.
Chatot, C.L., Ziomek, C.A., Bavister, B.L., Lewis, J.L. and Torres, I. (1989) An improved culture medium supports development of random-bred 1-cell mouse embryosin vitro.J. Reprod. Fertil. 86, 679–88.
Covarrubias, L., Nishida, Y. and Mintz, B. (1986) Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain.Proc. Natl Acad. Sci. USA 83, 6020–4.
Covarrubias, L., Nishida, Y., Terao, M., D-Eustachio, P. and Mintz, B. (1987) Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.Mol. Cell. Biol. 7, 2243–7.
Frels, W.I., Bluestone, J.A., Hodes, R.J., Capecchi, M.R., and Singer, D.S. (1985) Expression of a microinjected porcine class I major histocompatibility complex gene in transgenic mice.Science 228, 577–80.
Gordon, J.W., Chesa, P.G., Nishimura, H., Rettig, W.J., Maccari, J.E., Endo, T., Servalli, E., Seki, T. and Silver, J. (1987) Regulation of Thy-1 gene expression in transgenic mice.Cell 50, 445–52.
Hogan, B., Costantini, F. and Lacy, E. (1986)Manipulating the Mouse Embryo: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Marmur, J. (1961) A procedure for the isolation of DNA from microorganisms.J. Mol Biol. 3, 208–18.
Pivco, J., Grafenau, P., Oberfranc, M., Bulla, J., Kubovicova, E., Laurincic, J. and Hyttel, P. (1992) Recovery, structure and viability of porcine zygotes and 2-cell stages subjected to centrifugation.12th International Congress on Animal Reproduction and Artificial Insemination 2, 736–38.
Richa, J. and Lo, C.W. (1990) Embryo transfer into pregnant mice: An alternative to pseudopregnancy.Lab. Anim. Sci. 40, 637–8.
Saiki, R.K., Walsh, P.S., Levenson, C.H. and Erlich, H.A. (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.Proc. Natl Acad. Sci. USA 86, 6230–4.
SAS Institute, Inc. (1985) SAS/STAT Guide for personal computers. Cary, NC.
Velander, W.H., Page, R.L., Morcol, T., Russell, C.G., Canseco, R., Young, J.M., Drohan, W.N., Gwazdauskas, F.C., Wilkins, T.D. and Johnson, J.L. (1992) Production of biologically active human protein C in the milk of transgenic mice.Ann. N. Y. Acad. Sci. 665, 391–403.
Voss, A.K., Sandmöller, A., Suske, G., Strojek, R.M., Beato, M. and Hahn, J. (1990) A comparison of mouse and rabbit embryos for the production of transgenic animals by pronuclear microinjection.Theriogenology 34, 813–24.
Wagner, E.F., Covarrubias, L., Stewart, T.A. and Mintz, B. (1983) Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germline.Cell 35, 647–55.
Wall, R.J., Pursel, V.G., Hammer, R.E. and Brinster, R.L. (1985) Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei.Biol. Reprod. 32, 645–51.
Whitelaw, C.B.A., Springbett, A.J., Webster, J. and Clark, J. (1993) The majority of G0 transgenic mice are derived from mosaic embryos.Transgenic Res. 2, 29–32.
Wilkie, T., Brinster, R.L. and Palmiter, R.D. (1986) Germline and somatic mosaicism in transgenic mice.Dev. Biol. 118, 9–18.
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Canseco, R.S., Sparks, A.E.T., Page, R.L. et al. Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice. Transgenic Research 3, 20–25 (1994). https://doi.org/10.1007/BF01976023
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DOI: https://doi.org/10.1007/BF01976023