Am J Stem Cell 2012;1(2):163-173

Original Article
Successful derivation of EGFP-transgenic embryonic stem cell line from a
genetically non-permissive FVB/N mouse

Gurbind Singh, Tulasigeri M Totiger, Polani B Seshagiri

Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore-560 012, India

Received May 7, 2012; accepted May 29, 2012; Epub June 3, 2012; Published June 30, 2012

Abstract: Derivation of embryonic stem (ES)-cell lines from genetically non-permissive mouse strains, such as FVB/N, has been difficult,
despite this strain offering advantages for mouse transgenesis for developmental studies. We earlier generated β-actin promoter-driven
enhanced green fluorescent protein (EGFP)-transgenic FVB/N mice, expressing EGFP in all cells. Here, by optimizing culture system and
using RESGROTM ES-cell culture medium, we successfully derived EGFP-transgenic ES-cell line, ‘GS-2’ line, from F1 hybrid
blastocysts, from wild-type 129/SvJ female X EGFP-transgenic homozygous FVB/N male. The GS-2 ES-cell line exhibited all defining
criteria of a typical ES-cell line, including normal colony morphology and karyotype (40,XY), high replication-expansion efficiency
(passages: >100), expression of pluripotent markers (Oct-4, Nanog, Sox-2, SSEA-1 and others) and, embryoid body (EB) development
and EB differentiation to ecto-/meso-/endo-dermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively. GS-2 ES-cells
formed (i) teratoma containing three germ lineage-derived cell types, (ii) chimeric blastocysts and fetuses, following their aggregation
with wild-type 8-cell embryos, (iii) functional cardiac clusters and (iv) predominantly neural cell types when EBs were developed in KOSR-
supplemented medium. Taken together, we derived a robust EGFP-transgenic GS-2 ES-cell line, from a non-permissive transgenic
(FVB/N) mouse by a single cross to 129/SvJ wild-type mouse. The GS-2 ES-cell line exhibited full differentiation potential, in vitro/in vivo,
providing enormous opportunity for stem cell research, including experimental cell transplantation studies. (AJSC1205002).

Keywords: Embryonic stem cells, non-permissive mouse strain, transgenic ES-cell line, differentiation, cardiogenesis

Address all correspondence to:
Dr. Polani B Seshagiri
Department of Molecular Reproduction
Development and Genetics
Indian Institute of Science, Bangalore 560012, India.
Tel: 080-2293-2687; Fax: 080-23604312
AJSC Copyright © 2011-present. All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711, USA