| Embryonic stem cells (Embryonic Stem Cells, ESCs) can be carried out in vitro unlimited self-renewal, and meanwhile Embryonic stem cells ESCs can maintain the ability to differentiate into various cell types of the organism, and therefore embryonic stem cells ESCs in the field of regenerative medicine has broad application prospects.Preliminary experiments we have found that transcription factor Tfcp211 is a key downstream target genes of the LIF/STAT3 signaling pathway, changing Tfcp211 expression levels can greatly affect the LIF/STAT3 signaling pathway to promote self-renewal of mouse ESCs function, but the role of Tfcp211 specific molecular mechanisms have not been reported at home and abroad. Whether there is a critical and new transcription factor for mESCs (mouse embryonic stem cell) self-renewal.Up to now, it has been disclosed in numerous downstream target genes of STAT3, only down Tfcp211 expression, that it can weaken the LIF/STAT3 signaling promotes mESCs self-renewal function, thereby inducing cell differentiation, And so far identified genes that can induce mESCs differentiation are not many. Based on the important role of ESCs in the self update of Tfcp211, combined with a variety of efficient gene editing tools, such as lentivirus expression system and PiggyBac Transposon systems,it is possible to discover new genes that are essential for maintaining the undifferentiated state of mESCs. This paper intends to use gene expression and interference technology to screen and identify the Tfcp211 to promote the interaction of self-renewal ESCs gene in mice, expect to find maintain the undifferentiated state of mouse ESCs new regulatory factors or mechanisms. This paper selected MTA1, MTA2, MTA3 as our target genes to study the mechanism of their interaction with Tfcp211 to promote the self-renewal of mouse embryonic stem cells.The specific experimental program flow is as follows:First, is the culture of 46C mESCs, This paper used mouse embryonic stem cells 46C mESCs, provided by the Anhui university Medical Center Stem Cell.Its main process is divided into the following phases:medium configuration,cell recovery, frozen cells, gelatin-coated, cell passage.Secondly, we constructed the Tfcp211 overexpressing 46C mESCs, mainly divided into the following process:First Construction PB-Tfcp211 Vector:Design Tfcp211 primers, to obtain the target gene Tfcp211, target gene Tfcp211 connects PB plasmid. Recombinant Tfcp211 and PB plasmids were constructed and transformed into DH5acompetent cells. Selected monoclonal spread culture extract PB-Tfcp211 plasmid. The recombinant plasmid of Tfcp211 and PB was sequenced correctly, and 46C mESCs were transfected with PB-Tfcp211 recombinant plasmids, use the liposomes transfection. At this time over expression of Tfcp21146C mESCs construction completed, we identified Tfcp211 into 46C mESCs over expression.We use the AP alkaline phosphatase dyeing and WesternBlot identified the PB-Tfcp21146C mESCs overexpressing.After the Tfcp211 of 46C mESCs overexpress was constructed successfully, we’ll build PB-Tfcp21146C mESCs monoclonal cell lines.Then identify the PB-Tfcp21146C mESCs overexpressing ability of monoclonal cell lines.We use alkaline phosphatase dyeing and qRT-PCR identifying the PB-Tfcp21146C mESCs monoclonal cell lines overexpressing ability.All of the experiments above are related to the change of Tfcp211 expression to research the related function of ESCs self renewal in mice. After that we select the target gene MTA1, MTA2, MTA3 three as our target gene to study with Tfcp211 promote self-renewal of 46C mESCs related mechanisms.We used RNA interference technology interference MTA1, MTA2, MTA3 three, reduced the expression of MTA1, MTA2, MTA in mouse embryonic stem cells, observed their self-renewal capacity of stem cells in mouse embryos. In this case the use of mouse embryonic stem cells are 46C mESCs overexpressing Fcp211 monoclonal cell lines. First constructed MTA1, MTA2, MTA3 of shRNA lentiviral vectors:For Mtal, Mta2, Mta33 genes,design RNA interference primers, digested connection construct Mtal, Mta2, Mta3 three gene recombinant plasmids.After transforming the recombinant plasmid to DH5a competent cell,picked monoclones, after the expansion for restructuring Plko.1-shRNA Plasmid, extracted Plko.1-shRNA plasmid and used sequencing right Plko.1-shRNA plasmids for the next viral packaging. Virus package using Plko.1-shRNA plasmids and using 293FT cells.293FT cell culture method is basically the same as the method of culturing mouse ES cells.Transfection also used lipofectamine LTX liposomes. After the virus collection, infected Tfcp211 overexpressed 46C mESCs. After the re-use of AP alkaline phosphatase dyeing and qRT-PCR identified Tfcp11 overexpressing for transferred into Plko.1-shRNA 46C mESCs.under the premise of expression Tfcp211, the interference of the MTA1, MTA2, MTA3 three that can cause what effects on the self-renewal of 46C mESC.Finally, using immunoprecipitation technique to identify the Tfcp211 and MTA1 interaction in mouse embryonic stem cells.The entire experimental program processes described above, the main body is divided into two parts, the first part is Tfcp211 overexpression of mouse embryonic stem cells, the result is to promote 46C mESCs self-renewal. The second part is to use the first part of the over-expressed under Tfcp211 premise, RNA interference MTA1, MTA2, MTA3 to reduce their expression, observed mouse embryonic stem cell self-renewal if has any changes. As a result, only under the condition of interference MTA1 expression of mouse embryonic stem cell differentiated obviously that their self-renewal capacity declined.qRT-PCR results identified as follows:We have interfered with MTA1 mouse embryonic stem cells for qRT-PCR, where in the interference MTA146C mESCs and normal 46C mESCs, the mouse embryonic stem cells self-renewal related genes:Nanog, Esrrb and Rex1.The expression levels of Nanog, Esrrb and Rexl were significantly different.The expression levels of Nanog, Esrrb and Rexl were decreased in the interfered MTA1 with the 46C mESCs. That indicated interferencing MTA1 can make self-renewal of mouse embryonic stem cells was significantly reduced. In contrast, embryonic stem cell related self differentiation gene sox17 and Gata6 expression also significantly different, the expression levels of sox17 and Gata6 were increased in the interfered MTA1 with the 46C mESCs. Interferencing MTA1 can make 46C mESCs self differentiation significantly improved. Based on the above analysis, interference MTA1 can make 46C mESCs self-renewal ability decreased and to improve the differentiation ability, indicating MTA1 promoting embryonic stem cell self-renewal capacity. However, how those act between MAT1 and Tfcp2112 specific interactions remains to be further studied.The results of immunoprecipitation showed Tfcp211 and MTA1 in mouse embryonic stem cell interaction. |
