Microinjection of bovine embryos with a foreign gene and its detection at the blastocyst stage.
This study was designed to measure the survival and development of pronuclear stage bovine zygotes after microinjection with DNA and either culture in vitro or incubation in the sheep oviduct and to determine the percentage of embryos that retain exogenous DNA at the blastocyst stage. In vitro matured and fertilized oocytes were examined for pronuclear development 18 to 20 h after coincubation with sperm. An ovine somatotropin gene construct was microinjected into ova with visible pronuclei. Microinjected ova and unmanipulated ova from the same in vitro derived pool were cocultured with oviductal epithelial cells or incubated in the sheep oviduct and cocultured, respectively, for 7 d. Blastocysts were subjected to polymerase chain reaction analysis for detection of exogenous DNA. The percentage of cleaved embryos that formed blastocysts was similar for microinjected and unmanipulated cultured ova. The percentage of blastocysts recovered from the sheep oviduct that hatched was higher than for blastocysts that developed in coculture. Fourteen of 26 (54%) blastocysts that developed from microinjected ova were positive for the exogenous DNA by polymerase chain reaction. The implications are discussed for polymerase chain reaction detection of the exogenous gene construct in a relatively high proportion of microinjected blastocysts.
Behboodi
E
E
Department of Animal Science, University of California, Davis 95616-8521.
Anderson
G B
GB
Horvat
S
S
Medrano
J F
JF
Murray
J D
JD
Rowe
J D
JD
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001755:Blastocyst / Q000737:chemistry*; D002417:Cattle / Q000196:embryology*; D004247:DNA / Q000032:analysis*; D005307:Fertilization in Vitro; D018014:Gene Transfer Techniques; D013006:Growth Hormone / Q000235:genetics; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes; D016133:Polymerase Chain Reaction
D013006:Growth Hormone; D004247:DNA