Optimization Of An In Vitro Culture System For Rabbit ES-like Cells

The rabbit, as an animal model, has less research on the culture systemof ES cells. The exprement is to explore the optimal solution of rabbit ES cells invitro culture on the base of study other animal ES cells. Experiments were performedto compare with the effect of treatment for embryos and components of culturemedium and densities of feeder layer on the isolation and cultivation of rabbit es-likecells. The results obtained were as follows:1. Treated rabbit blastocysts with different methods. The result showed, theblastocysts in different condition had different cultured effects. The rabbit blastocystsculturing directly had bagan to attached after8~9d cultured in vitro, the ICM couldnot proliferate. the embryos which were treated with Pricking zona pellucida couldhatch out completely and begin to attach after72h, ICM formation rate and the rabbitES-like cells clone rate were higher than the other two groups(P<0.05), and the stemcells obtained were up to the7th passages.2. Exploring the efect of superovulation on developmental potential of rabbitblastocysts in vitro.96hour-aged rabbit blastocysts were collected-from naturalintercourse rabbits and superovulation-treated rabbits, the blastocysts were cultivatedon mouse fibroblast cell layers in vitro, compared with the natural intercourse group.Culture. The results indicated that superovulation attachment rate of blastulas、ICMformation rate and clone rate of F1generation of rabbit ES-like cells as well assubsequent were not significantly different (P>0.05). This study showed thatsuperovulation had little impact on developmental potential of the selected rabbitblastocysts in vitro.3. The research compared the effect of different culture medium on cloning andpassaging of rabbit blastocysts. The results showed: the time of three groups rabbitembryonic attachment were about72h. Rabbit embryonic attachment and ICM clonerate and the formation rate of F1cultured in serous medium were significantly higherthan another two groups(P<0.05). However, the equlity mixed medium of serum andKSR can better maintain the shape of ES cell-like line clone of rabbit and possesed high passages in subsequent passages.4. Axploring the effects of different densities of mouse embryonic fibroblast cell onisolation and culturing of rabbit ES-like cells, rabbit blastulas were cultured atdifferent densities(10×103· cm-2,20×103· cm-2, and40×103· cm-2) of feeder layer,The results showed that the cell clone rate of F1、F2generation of rabbit ES-like cellsat a density of20×103· cm-2feeder layer were significantly higher than other twogroups (P<0.05), and the passage result were the best.5. When isolated rabbit ESC were examined, the results showed: AKP stain andOct-4、Nanog immunohistochemisty stain of ES colonies were strongly positive.RNA of ES cell colonies were extracted, the size of Purpose fragment were consistentas expected By RT-PCR.So we obtained enough embryos by superovulation, and we pricked zonapellucida, put them in the medium of mixed in equal proportions FBS and KSRmedium in cultivation, we cultured rabbit ES-like cells on the MEF feeder layer at adensity of20×103· cm-2.