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

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 αS1‐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. © 1996 Wiley‐Liss, Inc.