Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos.
The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta-galactosidase (beta-gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were co-injected with the scaffold attachment sequences--SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta-galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta-galactosidase expression. The percentage of transgenesis with pCMV-lacZalone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.
Rosochacki
Stanisław Józef
SJ
Molecular Cytogenetic Department, Polish Academy of Sciences, Institute of Genetics and Animal Breeding, Jastrzebiec, 05-552 Wólka Kosowska, Poland. stanislawrosochacki@yahoo.com
Strzelecka
Małgorzata
M
Kozikova
Larysa
L
Matejczyk
Marzena
M
Obłap
Rusłan
R
Połoszynowicz
Jarosław
J
Zwierzchowski
Lech
L
D000818:Animals; D030801:Animals, Genetically Modified; D002970:Cleavage Stage, Ovum / Q000166:cytology / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D008845:Microinjections; D009030:Mosaicism / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011817:Rabbits; D011995:Recombination, Genetic / Q000235:genetics; D013997:Time Factors