Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction

Abstract

We have developed a simple digestion-polymerase chain reaction (PCR) assay for a simultaneous transgene detection and sexing of pronucleus-injected bovine preimplantation embryos. Bovine embryos were microinjected with dam-methylated gene construct and cultured in vitro for 6-7 days after the injections. The developed blastocysts and compact morulae were bisected and the embryonic biopsies representing mainly trophoblasts were subjected to the digestion-PCR, while the biopsied embryos remained in culture. Embryonic DNA was released with proteinase K and the samples were digested with a Dpnl-Bal31 mixture before the PCR amplification of the transgene, bovine alpha S1-casein, and bovine Y-chromosome fragments in the same reaction. The whole assay from biopsy to electrophoresis took less than 6 hr. The digestion removed up to 50 fg of dam-methylated transgene copies (unintegrated or contaminants) and also a few hundred copies of contaminating PCR products from the embryonic samples. The digestion-PCR assay eliminated all transgene contaminations from noninjected blastocysts, which were exposed to the microinjection DNA during the stay in injection chambers, and reduced the amount of transgene-positive embryos among pronucleus-injected blastocysts as compared with unmodified PCR. Analysis of 486 microinjected bovine embryo biopsies in 13 separate experiments revealed that we were able to sex 398 (82%) of the biopsies and 77 (19%) of the biopsies were scored as transgene positive and 57 (14%) as transgene questionable. Upon reanalysis of 41 of the biopsied embryos, 38 (93%) of the embryos were observed to be transgene negative and 2 questionable in both assays and uneven distribution of transgene copies was observed in one embryo. The results from sexing were in accordance with biopsies and remaining embryos in 38 (93%) of the embryos.