Nuclear transfer of adult and genetically modified fetal cells of the rat.
The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4-28%). This type of activation was also effective in reestablishing cleavage capability (19-71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14-16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16-28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos (n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production.
Hayes
E
E
Centre For Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia. eric.hayes@med.monash.edu.au
Galea
S
S
Verkuylen
A
A
Pera
M
M
Morrison
J
J
Lacham-Kaplan
O
O
Trounson
A
A
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology*; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002478:Cells, Cultured; D004171:Diploidy; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D005314:Embryonic and Fetal Development; D005260:Female; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000637:transplantation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008677:Metaphase; D051379:Mice; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000254:growth & development* / Q000648:ultrastructure; D010312:Parthenogenesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase