Production of Bovine Transgenic Conceptus; Possible Selection of Transgenic Embryos by Polymerase Chain Reaction.

Abstract

This study was conducted to examine the possibility of selection of transgenic bovine embryos by use of polymerase chain reaction (PCR) following microinjection of exogenous DNA before embryos were transferred to recipient cattle. DNA of bovine α-lactalbumin (α-LA) gene was microinjected into pronuclei of bovine embryos derived from in vitro maturation and fertilization. They were then cultured for 7 to 8 days to the blastocyst stage. To examine persistence of injected DNA during embryo development, embryos were analyzed by PCR at 0, 1, 3, 5 and 7 days following DNA microinjection. To distinguish the injected α-LA from the endogenous gene, gene-gene junction regions of head-to-tail concatemers of the injected DNA, which were formed when linearized DNA were injected into cell nuclei, were amplified by PCR. Injected DNA persisted at high rates (72 to 80%) until 5 days following DNA injection, but the detection rate at the blastocyst stage (20%) was lower than earlier stage (P<0.05), indicating that injected DNA into bovine embryos drastically decreased at the blastocyst stage. To determine if the PCR signals reflect transgenic status, biopsy samples of DNA-injected blastocysts were analyzed. Total of 1, 583 embryos were microinjected, and 124 (8.9%) developed to blastocysts. Twelve (11%) out of 109 samples biopsied from microinjected blastocysts were positive by PCR analysis. Eight PCR-positive embryos were transferred to 8 recipients. Also, 31 negative embryos were transferred to recipient heifers. Four out of 8 recipients that received PCR-positive embryos were pregnant and fetuses and placentae were recovered at 60 to 75 days of pregnancy. One of the samples, of which only a placenta was recovered, was found to be transgenic by both PCR analysis and Southern hybridization. The other samples had no injected DNA. On the other hand, 14 fetuses with placentae were recovered from recipients that received PCR-negative embryos. None of these samples were found to carry injected DNA. Our system could not detect single, or head-to-head- or tail-to-tail-joining transgenes; however, the results described here imply selective transfer of potential transgenic embryos to recipient heifers by the PCR selection.