The Production Of Single-cell Clone From Chicken Embryonic Stem Cells And Exogenous Gene Integrated SSCs By SB Transposon

Embryonic stem cells (ES cells) were isolated from animal inner cell mass (ICM) and cultured in vitro to establish cell clone lines. ES cells are topitent cells and have differentiation ability similar to embryo, they can be cultured and generated in vitro and maintained in undifferentiated states, also have the ability to form chimera. The origin of single-cell clone line is the same ancester cell, so there is strong homology, no genetic diversity, uniform biotic character and stationary cells line. This study is to compare the method of the preparation of chicken's stageâ…©ES cell's single-cell clone. The results of this study provide valuble reference for ES cell's single-cell clone, The main results were as follows: Single-cells were derived from the 3th passage multiplied clonal ES of stageâ…©, which were digested with 0.25% trypsin-EDTA.We use three different methods, cell limiting dilution method, Mouth sucker method and clone link method to pick up single cell and seed into individual well of a 96-well plate, and every well contained one cell. We detect the cell's special markers by cytochemistry and histoimmunochemistry. The results indicated that there is no one clone formed by Mouth sucker method, 1 by clone link method and 4 by cell limiting dilution method. We conclusioned that cell limiting dilution method is the easeat and it COS-â… t less time. We can use cell limiting dilution method to produce single-cell clonal.The 5 clones expressed a series of surface markers, such as: alkaline phosphotase, stage-specific embryonic antigen (SSEA)-1.The Sleeping Beauty (SB) transposon is one of the Tc1/mariner transposon families. The study showed that SB transposon was the highest transposition activity in vertebrates. In this study, pEGFP-N1 and PT2/BH carrier plasmid was used to construct PT-EGFP plasmid. After make sure PT-EGFP plasmid is exactitude by PCR and digesting by incision enzyme, we abstract and depurate the recombinant plasmid.Transfect COS-â… cells, observe in fluorescence microscope, then transfect Chicken Spermatogonial Stem Cells by electroporation with PT-EGFP and SB plasmid.This study mainly investigate the efficiency of exogenous gene integration encouraged by SB transposon plasmid of different density. We take the SB and PT-EGFP plasmid quality proportion fractionate gradient of 0:1,1:3,1:5,1:10,1:15,1:20,1:25, then transfect Chicken Spermatogonial Stem Cells by electroporation.The result of the experiment shows origin generation have the highest fluorescent light expression rate in quality proportion of 1:5,1:10,1:15 , the rate is 23.23%,24.25%,22.51%;after one passage , quality proportion of 1:5,1:10 have the highest,they are 4.67%,4.66%;after two passage quality proportion of 1:5,1:10,1:15 have the highest efficiency ,they are 0.94%,1.08%,0.94%. The main results indicate that SB plasmid have the efficiency of exogenous gene integration and quality proportion of 1:5,1:10,1:15 have the highest efficiency. Extract the DNA of second filial generation cells after trasfection, the result is masculine by PCR.This research may play a important role to make the Sleeping Beauty transposon used in chicken's transgene.