Isolation And Clone Of Rabbit Embryonic Stem Cell

Experiments were pertbrrned to determine the effect factors of culture. clone. isolation and passage of embryonic stern (ES) cell and embryonic germ (EG) cell in rabbit. Eight rabbit stem like stem cell lines were isolated by culture system composed of embryonic fibroblast feeder of Kun Ming strain white mouse(MEF), rnedium( low glucose DMEM, 1O%NBS, 1O%FCS,O.ImM P - mercaptoethunol, I OOlUIrnl penicilin, I OOlU/ml streptomycin sulfate) supplied with lOng/mi SCF and lOng/rn! LIF. The rabbit ES like cell lines were cloned to 4 passage, including 43 first ES like colonies (39.8%), 8 second passage ES like colonies (7.4%) ,3 third passage ES like colonies (2.7%) and I forth passage ES like colony(O.9%) In suspension culture and differentiation experiment in vitro, rabbit ES like cell formed embryonic like body. When ES like cells were cultured on degenerative feeder, they differentiated multiple types of cells, such as cardiac muscle and neurons. The effect of embryonic age. growth factor and feeder on isolation rabbit ES like cell was studied, the result showed: firstly, rabbit embryonic attachment and 1CM clo,ie rate were 72h(68.l%,50.O%), 96h(71.6%,53.6%), 120h(77.4%,45.2%). SCF and LIF can increase the rate of rabbit embryonic attachment(71.4%:34.8%). proliferation of 1CM cells and the number of ES like cell colonies during subculture. The feeder of MEF was better than the feeder of REF. The growth and developmental behavior between rabbit and mouse embryos was compared in ES culture system, (I) the trophablast cells of mouse embryo pushed the feeder cells away, and spread at the bottom of the dish. Most of the trophablast cells of rabbit embryo could not push the feeder away, but speed onto the feeder foriiiing a disc of embryo. (2) The mouse ICMs grew as clumps, while the rabbit embryo grew in different fashions, some grew as clump, some grew as migration clump, some grew as monolayer. (3) The 1CM of mouse was passaged after 4--id culturing. while the 1CM of rabbit was passaged after 3d culturing. Primordial germ cells (PGC) were isolated from 14?8d rabbit embryonic genital ridges and cocuitured on hornogenous embryonic fibroblast.The medium was low glucose DMEM, supplied with lO%NBS IO%FCS 0.1mM P - rnercaptoethuno!. I OOIU/rnl penicilin I OOlU/ml streptomycin sulfate I Ong/ml SCF and lOng/mI LIF. The rabbit EG cell lines derived from 14d PGC were cloned to 4 passage, l6d PGC were cloned to 2 passage, 18d PGCs have noEG clone.EG cells shared many characteristics with mouse ES cell: morphology of stem cell, colonial growth, maintenance of an undifferentiated state through serial passage, ability to form embryonic bodies in suspension culture and multiple type of differentiated cells in monolayer culture.