Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals.
The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10(-9) to 10(-5) U/nucleus, the amount of 5x10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI-Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.
Seo
B B
BB
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 113-8657, Tokyo, Japan.
Kim
C H
CH
Yamanouchi
K
K
Takahashi
M
M
Sawasaki
T
T
Tachi
C
C
Tojo
H
H
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects*; D046508:Culture Techniques; D004247:DNA / Q000008:administration & dosage*; D015246:Deoxyribonuclease EcoRI / Q000008:administration & dosage*; D004587:Electrophoresis, Agar Gel; D004622:Embryo, Mammalian / Q000502:physiology / Q000648:ultrastructure; D005260:Female; D018014:Gene Transfer Techniques; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D008845:Microinjections; D016133:Polymerase Chain Reaction
D004247:DNA; D015246:Deoxyribonuclease EcoRI