Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA.
We established a rapid procedure for obtaining transgenic mice by directly injecting an enhanced green fluorescent protein (EGFP)-expressing plasmid (pIRES-EGFP) into the ovaries of fertile mice. The frequency of transgenic mouse production was determined by pair-mating, and by polymerase chain reaction (PCR) and sequence analysis of DNA taken from the tails of the offspring. The mice that received the EGFP gene transmitted it to their offspring (F(1)). Genetic and PCR analyses of F(1) progeny confirmed that the inserted EGFP was stably inherited. Of six female F(1) mice, all were able to pass the foreign DNA on to the next generation (F(2)). In situ hybridization using paraffin-embedded sections of ovarian and testicular tissues from the F(1) and F(2) progeny showed that the introduced gene was expressed in the gonads of the animals. The chromosomal location of the injected DNA was determined by fluorescence in situ hybridization, and the frequency of multiple site versus single site insertions is 85.71% (18/21) analyzed by FISH. We anticipate great progress in murine genetic engineering using this technique.
Yang
Shu-Yan
SY
Laboratory of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
Wang
Ji-Gui
JG
Cui
Huan-Xian
HX
Sun
Shao-Guang
SG
Li
Quan
Q
Gu
Ling
L
Hong
Yan
Y
Liu
Peng-Peng
PP
Liu
Wei-Quan
WQ
D000818:Animals; D033481:Chromosomes, Mammalian / Q000235:genetics; D004247:DNA / Q000008:administration & dosage*; D004622:Embryo, Mammalian; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010053:Ovary; D010957:Plasmids / Q000008:administration & dosage*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA