Reprogramming Of Goat Fibroblasts To Pluripotent Stem Cells Using MRNA Of Four Transcription Factors

The somatic cells can be reprogramed into induced pluripotent stem cells by pluripotency transcription factors. So far, iPS cells have been successful for people, cattle, goat, sheep, mice, rat, monkey, rabbit, pig and other species. IPS cells have similar to embryonic stem cells. The technology provides a viable alternative for embryonic stem cell lines of hard to build up the cell lines. however, the traditional way of viral vector will be specific induction factors Oct4, Sox2, Klf4, and c-myc into cells, viruses can’t achieve the transfection efficiency of100%, obtain four factor at the same time expressing cell proportion is low, and the virus carrier will be randomly inserted into the genome of somatic cell, could lead to a normal gene expression of the affected, or cause gene mutations and activates protocarcinogenic gene expression. Plasmid vector, multiple protein expression systems, chemical although small molecules as well as translocation system as much as possible to reduce the exogenous gene and viral components of integrated, secure the safety of iPS cells get improved, but due to the complexity of operation, this paper analyzes the reasons of hard times, the preparation of low efficiency of restricts the development of iPS cell preparation technology. Compared with the above preparation technology of iPS cells, the mRNA induced technology in the theoretical advantage: on the one hand, avoid the risk of exogenous genes into somatic genome; On the other hand the induction efficiency than the above methods, the speed of cell reprogramming doubled. This study used Oct4, Sox2, Klf4, and c-Myc mRNA induced technology successfully get the iPS cells for goats. In addition, the caused by optimization of cultivation system and a goat iPS cells success to22generation. In this paper, the main research results are as follows:1. Cloning and analysis of four pluripotent transcription factors related to goat iPS cells Referring to the sequences that have been reported in GeneBank, goat Oct4、Sox2、Klf4and c-Myc (OSKC) were designed and synthesized specific primers. RT-PCR was used to get coding sequences (CDS) of OSKC from ridge, epidermis and small intestine of goat fetus. The results showed that, the CDS length of OSKC were1083bp、62bp、1434bp and1320bp, respectively. Homology analysis indicated that, the nucleotide sequences of goat OSKC were highly conservative with those of goat, sheep, cattle and pig. The specific homologies of these genes in these four species were showed as follow: Oct4,100%、100%、97%and94%; Sox2,100%、100%、98%and94%); Klf4,100%、100%、97%and94%); c-Myc,100%、100%、94%and91%. All these results indicated that the obtained cloning sequences of these four genes were correct.2. The construction of different pcDNA3.0eukaryotic expression vectors and transcription in vitroThe above OSKC genes and pcDNA3.0vector were treated with corresponding restriction enzymes for double enzyme, and then made connection transformation. Microbial PCR, single and double enzyme identification and sequencing were detected to confirm the four eukaryotic expression vectors, pcDNA3-oct4、pcDNA3-sox2、pcDNA3-klf4and pcDNA3-c-myc, were all restructured correctly. The mRNA concentration of OSKC transcription factors and EGFP were283.55ng/ul、274.81ng/ul、448.54ng/ul、295.73ng/ul and315.43ng/ul respectively; and the OD value were1.89、1.89、1.96、1.92and1.99respectively. All of the OSKC transcription factors and EGFP had an unique extremum of10mm absorbance value in the260nm wavelength. This indicated that the obtained mRNA of OSKC and EGFP were pure, and could meet the requirements of subsequent transfection experiment.3. Goats iPS induction and cultivation system optimizationThis article was to study the effects of different kinds of breeding layers and culture solutions on the obtaining and developing of goat iPS. The results showed that, with "MEF cells:GEF cells=1:1" feeding layer and " KnockoutDMEM+20%KSR " culture solution, the goat iPS got the best induction efficiency. Moreover, the cloning efficiency of F1, F2and F3generation of goat iPS were1.15%,63.21%,36.98%, which was also the highest rate among all the induction groups. Using morphological criteria to choose original generation clone expand training, a strain of goat iPS cells using the training system has been spread to22generation. original generation clone was chose with morphological criteria for enlage cultivation, one cell strain of goat iPS were passaged until22generation with the above culture system.4. The biological characteristics of goat iPSThe induced goat iPS of this study were showed colony growth, the uplift of each clone was like island; all the iPS cells were in high brightness, and the cell edge was neat, smooth and high refractive; closely packed iPS cells had a clear boundary with the layer cells. Goat iPS clone, similar to goat ESCs clone and mouse iPS clone, was positive for AP staining. RT-PCR results showed that, goat iPS cell lines expressed a variety of ES cell markers, including Oct4、Sox2、 Nanog、Daxl、Dnmt3b and Gdf3. The IF results showed that, goat iPS cell lines expressed a variety of ES cell markers, including Oct4、Sox2、Klf4、c-Myc、Nanog、Rex1、SSEA-1、 TRA-1-60and TRA-1-81. Karyotype analysis showed that goat iPS cells had normal karyotype, with58autosomes and2sex chromosomes. With culture in vitro, goat iPS could spontaneously differentiate to epithelioid-like cells, nerve-like cells and fat-like cells. Goat iPS could form embryoid body by uspension cultivation in vitro.5. The goat somatic cell reprogramming mechanisms under the mRNA inducedCell morphology test results showed that the goat somatic cell morphology changed after transfection; qRT-PCR results showed that endogenous Nanog, Tert genes expressions in GEF cell after transfected mRNA in12days, indicated endogenous regulation network mechanism activated, and began to express endogenous genes; Western blot analysis was similar to qRT-PCR results, showed that endogenous mechanism of regulation network in mRNA transfection12days activated.Above all, early reprogramming of somatic cells after transfection rely mainly on exogenous expression of transcription factors to drive, goat iPS cell pluripotency rely mainly on related to maintain the internal expression of transcription factors.