Germline Transmission of an Embryonic Stem Cell Line Derived from BALB/c Cataract Mice

Xinrong Peng,Qijun Qian,Yong Zhang,Wuyang Zhao,Liqing Zhang,Chuanyin Shi,Mengchao Wu,Lihua Jiang,Ying Wang,Tao Liu,Austin John Cooney

doi:10.1371/journal.pone.0090707

Abstract

Mice embryonic stem (ES) cells have enabled the generation of mouse strains with defined mutation(s) in their genome for putative disease loci analysis. In the study of cataract, the complex genetic background of this disease and lack of long-term self-renewal ES cells have hampered the functional researches of cataract-related genes. In this study, we aimed to establish ES cells from inherited cataract mice (BALB/CCat/Cat). Embryos of cataract mice were cultured in chemical-defined N2B27 medium with the presence of two small molecules PD0325901 and CHIR99021 (2i) and an ES cell line (named EH-BES) was successfully established. EH-BES showed long-term self-renewal in 2i medium and maintained capacity of germline transmission. Most importantly, the produced chimera and offspring developed congenital cataract as well. Flow cytometry assay revealed that EH-BES are homogeneous in expression of Oct4 and Rex1in 2i medium, which may account for their self-renewal ability. With long-term self-renewal ability and germline-competent, EH-BES cell line can facilitate genetic and functional researches of cataract-related genes and better address mechanisms of cataract.

Highlights

Cataract is the most common cause of blindness and the development of cataract can be triggered by a variety of reasons including aging, trauma, radiation, etc
To derive embryonic stem (ES) cells, embryos from BALB/CCat/Cat mice were cultured in 2i medium with the presence of 1 mM PD0325901 and 3 mM CHIR99021 as previously described [9] and results showed that undifferentiated ES-like colonies appeared in 3 days on gelatin-coated plastics (Fig. 1A)
We showed that EH-BES cells could maintain long-term self-renewal in chemical-defined medium in the absence of leukemia inhibitory factor (LIF) and feeder (Fig.1B), indicating that LIF is unnecessary for maintaining self-renewal of EH-BES cell line

Summary

Introduction

Cataract is the most common cause of blindness and the development of cataract can be triggered by a variety of reasons including aging, trauma, radiation, etc. Later emerged chemical-defined 2i medium [9] has enabled the derivation of ES cells from C57BL/6 mice, Kunming mice, and for the first time from rat [10,11,12]. It has not been proved whether ES cells from BALB/C hereditary cataract mice can maintain self-renewal and germline transmission ability in 2i medium

Objectives

Methods

Results

Conclusion