Establishment Of Embryonic Stem Cell Lines Of The Mouse Of Inherited Cataract

Congenital cataracts are one of the most significant causes of visual impairmentand blindness during infancy, with an estimated prevalence of1to6cases per10,000live births. All three types of Mendelian inheritance have been reported for cataract:autosomal dominant transmission, autosomal recessive transmission and X-linkedtransmission. Animal model have been mainly used by sciensits for cataractpathological research. Up to now, almost one hundred and forty mouse models ofinherited cataract have been found at home and abroad. They are mainly caused byspontaneous mutations and induced mutations, and some of them were established bytransgenic manipulation in knockout or transgenic mice. But no embryonic stem celllines of the mouse of inherited cataract have been reported.Embryonic stem (ES) cells are derived from the inner cell mass(ICM) ofpreimplantation blastocysts or primordial germ cells(PGC), which have thecharacteristic of self-renewal and multilineage differentiation potential. Since thesuccessful establishment of mouse embryonic stem cells by Evans and Kaufman in1981, the research for embryonic stem cells has been expanded both in breadth and indepth through these years. Because of their pluripotency and indefinite self-renewal,ESCs have tremendous potential for embryonic development research, transgenicmanipulation, pathological study, medicine test, animal model establishment, tissuegeneration, organ transplantation and other biological areas.In this experiment,3.5days blastocysts were cultured on mouse embryonicfibroblasts(MEFs) in the basic medium N2B27containing two inhibitors (2i):CHR99021and PD0325901, then Inner cell mass (ICM) were picked up and reseeded.After the ES-like colonies appeared, passaged them with2i medium to generatepermanent ES cell lines. Then we identified the morphology and karyotype of new EScell line, carried out other trials to comfirm the establishment of embryonic stem cell lines from the mouse model of inherited cataract, including the alkaline phosplmtase(AKP) activity, the expresstion of pluripotency markers, and analyze the capacity todifferentiate into derivatives of all three germ layers even to incorporate into thedeveloping embryo. We got six inherited BALB/cCat/CatES cell lines. All of them havethe typical colony with morphological characteristics of ES cells, and the ratioes ofnormal karyotype were80%. The testing results of Immunofluorescence staining,Western Blot, RT-PCR and qRT-PCR analysis, showed that the BALB/cCat/CatES celllines expressed strong AKP activity and SSEA-1，Oct4, Nanog, Sox2and so on. Theircapacity to form embryoid bodies (EBs) and teratoma demonstrated the pluripotentproperties of the cell lines. Four pups exhibited coat coloring indicative of thepresence of BALB/cCat/CatES cell, and three of them suffered from congenitalcataracts.The establishment of embryonic stem cell lines of the mouse of inherited cataract,the establishment of this model fills the gap in related field, benefits the research ofcatarac pathology and genetic therapy!...