Production of bovine transgenic conceptus; possible selection of transgenic embryos by polymerase chain reaction.

摘要

DNA of bovine alpha-lactalbumin (alpha-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 analysed by PCR at 0, 1, 3, 5 and 7 days following DNA microinjection. To distinguish the injected alpha-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 (72-80%) until 5 days following DNA injection, but the detection rate at the blastocyst stage (20%) was lower (P<0.05) than earlier stage, 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 analysed. A total of 1,583 embryoswere microinjected, and 124 (8.9%) developed to blastocysts. Twelve (11%) 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 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 imply selective transfer of potential transgenic embryos to recipient heifers by the PCR selection.