Abstract
The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days ofin vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocyst stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 μg ml−1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development. Also, unintegrated copies of the transgene probably exist at least until the blastocyst stage, and thereafter are degraded to the extent that they can no longer be detected by PCR.
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References
Aasen, E. and Medrano, J.F. (1990) Amplification of the ZFY and ZFX genes for sex identification in humans.Bio/Technology 8, 1279–81.
Birnbaum, N.C. and Doty, J. (1979). A rapid procedure for screening recombinant plasmid DNA.Nucl. Acids Res. 7, 1513–23.
Bishop, J.O. and Smith, P. (1989) Mechanism of chromosomal integration of microinjected DNA.Mol. Biol. Med. 6, 283–98.
Bradbury, M.W., Isola, L.M. and Gordon, J.W. (1990) Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos.Proc. Natl Acad. Sci. USA 87, 4053–7.
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 by microinjection.Proc. Natl Acad. Sci. USA 82, 4438–42.
Burdon, T.G. and Wall, R.J. (1992) The fate of microinjected genes in preimplantation mouse embryos.Mol. Reprod. Dev. 33, 436–42.
Campbell, S.M., Rosen, J.M., Hennighausen, L.G., Strech-Jurk, U. and Sippel, A.E. (1984) Comparison of the whey acidic protein genes of the rat and mouse.Nucl. Acids Res. 12, 9685–7.
Canseco, R.S., Sparks, A.E.T., Russell, C.G., Page, R.L., Johnson, J.L., Velander, W.H. and Gwazdauskas, F.C. (1994) Gene transfer efficiency during gestation and the influence of cotransfer of non-manipulated embryos on production of transgenic mice.Transgenic Res. 3, 20–5.
Chatot, C.L., Ziomek, C.A., Bavister, B.D., Lewis, J.L. and Torres, I. (1989) An improved culture medium supports development of random-bred 1-cell mouse embryosin vitro.J. Reprod. Fert. 86, 679–88.
Covarrubias, L., Nishida, Y. and Mintz, B. (1986) Early postimplantation embryo lethality due to DNA rearrangements in 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.
Fuller, D.T., Kochevar, D.T., Brumbaugh, G.W., Womak, J.E. and Kraemer, D.C. (1993) Pretreatment of murine embryos prior to the polymerase chain reaction.Theriogenology 39, 221.
Gardner, R.L. (1978) The relationship between cell lineage and differentiation in the early mouse embryo. In Gehring, W.J. ed.,Genetic Mosaics and Cell Differentiation, pp. 211–41. Berlin: Springer Verlag.
Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A. and Ruddle, F.H. (1980) Genetic transformation of mouse embryos by microinjection of purified DNA.Proc. Natl Acad. Sci. USA 77, 7380–4.
Hanley, T. and Merlie, J.P. (1991) Transgene detection in unpurified mouse tail DNA by polymerase chain reaction.Bio-Techniques 10, 56.
Hogan, B., Constantini, F., and Lacy, E. (1986)Manipulating the Mouse Embryo: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Holding, C. and Monk, M. (1989) Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos.Lancet (Sept. 2), 532–5.
Joshi, A.K., Baichwal, V. and Ames, G.F.L. (1991) Rapid polymerase chain reaction amplification using intact bacterial cells.Bio Techniques 10, 42–6.
King, D. and Wall, R.J. (1988) Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene.Mol. Reprod. Dev. 1, 57–62.
Kunieda, T., Xian, M., Kobayashi, E., Imamichi, T., Moriwaki, K. and Toyoda, Y. (1992) Sexing of mouse preimplantation embryos by detection of Y chromosome-specific sequences using polymerase chain reaction.Biol. Reprod. 46, 692–7.
Lin, F.L.M., Sperle, K. M. and Sternberg, N.T. (1987) Extrachromosomal recombination in mammalian cells as studied with single-and double-stranded DNA substrates.Mol. Cell. Biol. 7, 129–40.
Mercier, B., Gaucher, C., Feugeas, O.E. and Mazurier, C. (1990) Direct PCR from whole blood without DNA extraction.Nucl. Acids. Res. 18, 5908.
Nimomiya, T., Hoshi, M., Masaynagao, A. and Yuki, A. (1989) Selection of mouse preimplantation embryos carrying exogenous DNA by polymerase chain reaction.Mol. Reprod. Dev. 1, 1–7.
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.
Takeda, S. and Toyoda, Y. (1991) Expression of SV40-lacZ gene in mouse preimplantation embryos after pronuclear microinjection.Mol. Reprod. Dev. 30, 90–4.
Verlinsky, Y., Ginsburg, N., Lifchez, A., Valle, J., Moise, J. and Strom, C.M. (1990) Analysis of the first polar body: preconception genetic diagnosis.Human Reprod. 5, 826–9.
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.
Wu, G.Y. and Wu, C.H. (1987) Receptor-mediatedin vitro gene transformation by a soluble DNA carrier system.J. Biol. Chem. 262, 4429–32.
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Page, R.L., Canseco, R.S., Russell, C.G. et al. Transgene detection during early murine embryonic development after pronuclear microinjection. Transgenic Research 4, 12–17 (1995). https://doi.org/10.1007/BF01976496
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DOI: https://doi.org/10.1007/BF01976496