Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events.
There are several methods of modifying bovine genomes. Pronuclear microinjection is more widely used but it is still to be improved. Searches for alternatives have lead to the development of new methods including SMGT (Sperm Mediated Gene Transfer), in which live spermatozoa are used as vehicles for DNA delivery during in vitro fertilization. In previous studies, we presented evidence that a highly repetitive Alu-like repeat favours transgenesis by homologous recombination (HR). Up to 60% integration via HR was obtained following pronuclear microinjection of a Pst1 beta-actin GFP DNA construction. In the present study, we show that HR-mediated integration is also possible using SMGT, since bovine spermatozoa electroporated with the same DNA construct are able to transfer it to a high proportion of embryos obtained by in vitro fertilization. Swim-up selected bovine spermatozoa were mixed with the Pst1 beta-actin GFP construct (6 x 10(6) spermatozoids were incubated with 600 ng of muDNA), submitted or not to electroporation (300 V, 25 F) and treated or not with DNase I. The process of electroporation itself did not affect in vitro embryonic development. However, oocytes fertilized with electroporated DNA-treated spermatozoa developed beyond the 16-cell stage in proportions that were significantly lower (27% with Pst1 beta-actin GFP and 34% with beta-actin GFP) compared to the control without DNA (44%). On the other side, the use of electroporation significantly increased the uptake of DNA. The number of homologous recombination events detected by PCR went from 3.5% without electroporation to 46.5% after electroporation. In conclusion, our results confirm that spermatozoa electroporation combined with homologous recombination in a highly repetitive Pst1 sequence is a feasible method to obtain transgenic bovine embryos.
Rieth
A
A
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy (Québec), Canada.
Pothier
F
F
Sirard
M A
MA
D000818:Animals; D002417:Cattle; D004247:DNA; D018274:Electroporation / Q000379:methods; D005314:Embryonic and Fetal Development; D018014:Gene Transfer Techniques; D008297:Male; D009865:Oocytes / Q000502:physiology*; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*
D004247:DNA