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 OJAS  Vol.2 No.4 , October 2012
Murine embryo development following cytoplasmic injection of linear and condensed DNA
Abstract: In 1985 Brinster et al. [1] observed that linearized DNA injected in the cytoplasm of mouse zygotes underwent spontaneous supercoiling within 24 h. This finding suggests that DNA prefers and functions best in tertiary structure. In an effort to improve the efficiency of transgenesis by cytoplasmic injection, DNA was condensed with MgCl2to form a three dimensional rod-shaped DNA prior to injection in pronuclear stage murine zygotes. The DNA used was enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) and served as a marker for gene integration and protein expression in culture conditions. The condensed CMV-EGFP construct was injected in the cytoplasm at 3 concentrations (100, n = 816; 425, n = 464; and 625 μg/ml, n = 708). For comparison linear CMV-EGFP construct was injected into the pronucleus (5 μg/ml, n = 196) and into the cytoplasm (625 μg/ml, n = 628). In all treatment groups the control and buffer injected embryos developed similarly. Among DNA treatment groups, the highest development of fluorescing embryos was observed in zygotes injected in the cytoplasm with linear CMV-EGFP (625 μg/ml); however, zygotes injected in the cytoplasm with condensed CMV-EGFP (625 μg/ml) had the highest percentage (44%) of fluorescing embryos, the highest percentage (16.7%) of fluorescing morula and blastocysts, and the lowest percentage of fluorescence mosaicism at every stage of embryo development after 4 d in culture; thereby making it the best method for generating transgenic animals.
Cite this paper: Dunlap-Brown, M. , Butler, S. , Velander, W. and Gwazdauskas, F. (2012) Murine embryo development following cytoplasmic injection of linear and condensed DNA. Open Journal of Animal Sciences, 2, 244-252. doi: 10.4236/ojas.2012.24034.
References

[1]   Brinster, R.L., Howard, Y.C., Trumbauer, M.E., Yagle, M.K. and Palmiter, R.D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice by micro-injecting eggs. Proceedings of the National Academy of Sciences, 82, 4438-4442. doi:10.1073/pnas.82.13.4438

[2]   Page, R.L., Butler, S.P., Subramanian, A., Gwazdauskas, F.C., Johnson, J.L. and Velander, W.H. (1995) Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures. Transgenic Research, 4, 353-360. doi:10.1007/BF01973753

[3]   Widom, J. and Baldwin, R.L. (1980) Cation-induced toroidal condensation of DNA. Journal of Molecular Biology, 144, 431-453. doi:10.1016/0022-2836(80)90330-7

[4]   Ma, C. and Bloom-field, V.A. (1994) Condensation of supercoiled DNA induced by MnCl2. Biophysical Journal, 67, 1678-1681. doi:10.1016/S0006-3495(94)80641-1

[5]   Chauhan, M.S., Nadir, S., Bailey, T.L., Pryor, A.W., Butler, S.P., Notter, D.R., Velander, W. H. and Gwazdauskas, F.C. (1999) Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct. Journal of Dairy Science, 82, 918-926. doi:10.3168/jds.S0022-0302(99)75310-5

[6]   Bloomfield, V.A. (1991) Condensation of DNA by multi- valent cations: Considerations on mechanism. Biopolymers, 31, 1471-1481. doi:10.1002/bip.360311305

[7]   Hogan, B., Costantini, F. and Lacy, E. (1986). Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor Laboratory Press, New York.

[8]   Canseco, R.S., Sparks, A.E.T., Page, R.L., Russell, C.G., Johnson, J.L., Velander, W.H., Pearson, R.E., Drohan, W.N. and Gwazdauskas, F.C. (1994) 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. doi:10.1007/BF01976023

[9]   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 embryos in vitro. Journal of Reproduction and Fertilty, 86, 679- 688. doi:10.1530/jrf.0.0860679

[10]   SAS Institute, Inc. (1985) SAS user’s guide: Statistics. Cary, NC, USA.

[11]   Iqbal, K., Barg-Kues, B., Broll, S., Bode, J., Niemann, H. and Hues, W.A. (2009) Cytoplasmic injection of circular plasmids allows targeted expression in mammalian embryos. BioTechniques, 47, 959-968. doi:10.2144/000113270

[12]   Schmotzer, C.A., Butler, S.P., Pearson, R.E., Velander, W.H. and Gwazdauskas, F.C. (2003). Assessment of murine embryo development following cyto-plasmic microinjection of condensed DNA into murine embryos using electropulsation. Transgenics, 4, 55-63.

[13]   Yamauchi, Y., Doe, B., Ajduk, A. and Ward, M.A. (2007) Genomic DNA damage in mouse transgenesis. Biology of Reproduction, 77, 803-812. doi:10.1095/biolreprod.107.063040

[14]   Page, R.L., Canseco, R.S., Russell, C.G., Johnson, J.L., Velander, W.H. and Gwazdauskas, F.C. (1995) Transgene detection during early embryonic development after pronuclear microinjection. Transgenic Research, 4, 12-17. doi:10.1007/BF01976496

[15]   Perry, A.C., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyoda, Y. and Yanag-imachi, R. (1999) Mammalian transgenesis by intracytoplasmic sperm injection. Science, 284, 1180-1183. doi:10.1126/science.284.5417.1180

[16]   Szczygiel, M.A., Kusakabe, H., Yanagimachi, R. and Whittingham, D.G. (2002) Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa. Biology of Reproduction, 67, 1278-1284. doi:10.1095/biolreprod67.4.1278

[17]   Szczygiel, M.A., Moisyadi, S. and Ward, W.S. (2003) Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biology of Reproduction, 68, 1902-1910.

[18]   Covarrubias, L., Nishida, Y. and Mintz, B. (1986) Early postimplantation embryo lethality due to DNA rearrangements in transgenic mouse strain. Proceedings of the National Academy of Sciences USA, 83, 6020-6024. doi:10.1073/pnas.83.16.6020

[19]   Volgina, V.V., Khodarew, N.N., Volgin, A.Y., Votrin, I.I. and Pevnitskii, L.A. (1988) Comparative study of Ca, Mg-dependent endonuclease in cell nuclei by the use of monoclonal antibodies. Bulletin of Experimental Biology and Medicine, 105, 498-500. doi:10.1007/BF00841185

[20]   Burdon, T.G. and Wall, R.J. (1992) Fate of microinjected genes in preimplantation mouse embryos. Molecular Reproduction and Development, 33, 436-442. doi:10.1002/mrd.1080330410

[21]   Cousens, C., Carver, A.S., Wilmut, I., Colman, A., Garner, I. and O’Neil, G.T. (1994) Use of PCR-based methods for selection of integrated transgenes in preimplantation em- bryos. Molecular Reproduction and Development, 39, 384- 391. doi:10.1002/mrd.1080390406

[22]   Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y. and Okabe, M. (1995) A rapid and non-invasive selection of transgenic embryos before implanta- tion using green fluorescent protein (GFP). FEBS Letters, 375, 125-128. doi:10.1016/0014-5793(95)01162-8

[23]   Krisher, R.L., Gib-bons, J. R., Canseco, R.S., Johnson, J.L., Russell, C.G., Notter, D.R., Velander, W.H. and Gwaz- dauskas, F.C. (1994) Influence of time of gene microin- jection on development and DNA detection frequency in bovine embryos. Transgenic Research, 3, 226-231. doi:10.1007/BF02336775

[24]   Adenot, P.G., Mercier, Y., Renard, J. and Thompson, E.M. (1997) Differential H4 acetyla-tion of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos. Development, 124, 4615-4625.

[25]   Wu, G.Y. and Wu, C.H. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. Journal of Biological Chemistry, 262, 4429-4432.

[26]   Palmiter, R.D., Wilkie, T.M., Chen, H.Y. and Brinster, R.L. (1984) Transmission distortion and mosaicism in an unusual transgenic mouse pedigree. Cell, 36, 869-877. doi:10.1016/0092-8674(84)90036-9

[27]   Wall, R.J., Kerr, D.E. and Bondioli, K.R. (1997) Transgenic dairy cattle: Genetic engineering on a large scale. Journal of Dairy Science, 80, 2213-2224. doi:10.3168/jds.S0022-0302(97)76170-8

[28]   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 Research, 2, 29-32. doi:10.1007/BF01977678

[29]   Chan, A.W.S., Kukolj, G., Skalka, A.M. and Bremel, R.D. (1999) Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection. Molecular Reproduction and Development, 52, 406-413. doi:10.1002/(SICI)1098-2795(199904)52:4<406::AID-MRD9>3.0.CO;2-P

 
 
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