title: Embryo culture-based generation of enhanced green fluorescent protein-transgenic mice.
abstract: 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.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Vikram
authors.initials: V
authors.lastname: Devgan
authors.forename: Monzy
authors.initials: M
authors.lastname: Thomas
authors.forename: Kolathur S
authors.initials: KS
authors.lastname: Ullas
authors.forename: Manchanahalli R S
authors.initials: MR
authors.lastname: Rao
authors.forename: Polani B
authors.initials: PB
authors.lastname: Seshagiri
mesh_terms: 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
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA