Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection.
The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos.
Kaneko
Takehito
T
Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, HI 96822, USA.
Moisyadi
Stefan
S
Suganuma
Ryota
R
Hohn
Barbara
B
Yanagimachi
Ryuzo
R
Pelczar
Pawel
P
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002467:Cell Nucleus; D004277:DNA, Single-Stranded / Q000008:administration & dosage / Q000378:metabolism; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D045522:Recombinases / Q000008:administration & dosage* / Q000378:metabolism; D020554:Sperm Injections, Intracytoplasmic; D014162:Transfection / Q000379:methods
D004277:DNA, Single-Stranded; D045522:Recombinases; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate