Embryo culture-based generation of enhanced green fluorescent protein-transgenic mice.
One of the limitations of transgenesis is low efficiency. In this study, we generated transgenic mice harboring the enhanced green fluorescent protein (EGFP) gene, under the control of chicken-beta-actin promoter and cytomegalovirus enhancer, using two approaches and compared their efficiencies. One involved culture of EGFP-injected embryos developing through EGFP-expressing "green" blastocysts, followed by their transfer to uterus. The second was oviductal-transfer of EGFP-injected-eggs. Embryo culture-based-transgenesis (ECBT) produced 100% transgenic mice, unlike the second approach. Moreover, ECBT required reduced number of recipients and markedly increased pregnancy rates. Of the nine founders, seven exhibited ubiquitous EGFP-expression, one (GU1) was a mosaic and the other (G18) was non-expressing. The molecular basis for this was attributed to repeat-induced gene silencing, since the G18 had a high copy number (approximately 99/genome) of the non-mutated and non-rearranged EGFP-transgene integrated at a single site. Our results show the superiority of ECBT over the conventional oviductal approach for generating transgenic "green" mice.
Devgan
Vikram
V
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Thomas
Monzy
M
Ullas
Kolathur S
KS
Rao
Manchanahalli R S
MR
Seshagiri
Polani B
PB
D000818:Animals; D000831:Animals, Newborn; D001755:Blastocyst / Q000166:cytology; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000235:genetics; D004247:DNA / Q000008:administration & dosage; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D018628:Gene Dosage; D020868:Gene Silencing; D005838:Genotype; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011247:Pregnancy
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA