Islet-1's Indication Of C₃H₁₀T1/2 Cells Differentiate Into Cardiomyocyte-like Cells Specifically

ObjectiveTake advantage of lentiviral vector that could up-regulate and down-regulate the expression of Islet-1, to Investigate whether Islet-1 function as an promoter that makes C3H10T1/2 embryonic stem cell differentiate into cardiomyocyte-like cells; Clear-cut whether this function of Islet-1 is cardiac-specific.Methods and materials1. Construct lentiviral expression vectors: Islet-1 gene was obtained by PCR. Digested it and pLenO-WPI vector by restriction enzyme. Purify the products and then recombination them, and then transform them into Competent bacterial cells. Positive clones were indentified by PCR analysis. The forward and reward primer were designed targeting the vector and Islet-1 gene specially to make sure that we inserted the right gene. Sequencing the positive clones and compare it with Islet-1 gene and select the right ones as expression-vectors. Then infect positive colognes that we amplified into 293T along with the other 3 helper plasmids to produce lentiviral vector. 2. 3 groups of RNAi-target of mouse Islet-1 gene were designed, Corresponding ShRNA oligo(Sh1,Sh2 and Sh3)were synthesized and then inserted to the PLVTHM vector that digested by endonuclease. Agarose gel electrophoresis and sequencing were used to select and indentify positive clones. Extract positive clones and then mix them with E.coli to amplify positive clones. Then infect colognes that we amplified into 293T along with the other 3 helper plasmids to produce lentiviral vector.3. Transfect of C3H10T1/2: lentiviral vector(Moi=20) were co-cultured with C3H10T1/2(60% cell density) on the occasion of 8mg/L Polybrene. Real-Time fluorescent quantitative PCR and western-blot were used to test the expression of Islet-1.4. Function of Islet-1: There were two parts of this investigation, the positive part(expression group) and the negative part(Rnai group). Three groups were set:C3H10T1/2 group, blank control group, test group(Islet-1 expression group or Islet-1 RNAi group). RT-Q-PCR was used to test MEF-2c,GATA-4 and NKx2.5, which stands for cardiac transcription factor. Western-blot was used to determine the expression of cTnT. Meanwhile, Liver-specific gene and protein AFP and ALB, Bone-specific gene and BGP and BALP, Neural-specific gene and protein Nestin ,NSE and GFAP were checked use the same methods. RESULTS1. Fragment that we got after digest was about 2400bp identified by PCR. Gene sequencing showed we inserted the right segment.2. PCR evaluation showed we inserted a 330bp gene segment, which was the right size according to MluI and XbaI restriction enzyme cutting site. Gene sequencing showed improving result.3. GFP was detectable 4 days after cells were infected with lentivirus. RT-Q-PCR showed the Islet-1-expression group got a higher(P<0.05) level of Islet-1 gene expression while the RNAi-group got lower(P<0.05)compared to the blank control group. Western-blot showed Islet-1 protein expression in Islet-1-expression group. There were no expression of Islet-1 in RNAi-group, as while as in blank control group.4. The function of Islet-1 protein: In positive test(Islet-1 expression), TR-Q-PCR showed cardiac transcription factor MEF-2c,GATA-4 and NKx2.5 were up regulated after Islet-1 induction. Western-blot showed cTnT turned up and immunofluorescence showed it was expressed in kytoplasm. In negative test(RNAI of Islet-1), PCR and Western-blot showed no cardiac related gene or protein expression. Markers of liver and bone system that we selected got no expression in positive and negative group according to the results of PCR and Western-blot; NSE expressed in both test group and control groups and there were no difference between them. Conclusions1. As a transcription factor, Islet-1 could promote C3H10T1/2 embryonic stem cell differentiate into cardiomyocyte-like cells. Islet-1 started the expression of early cardiac transcription factor, resulted in the appearance of cardiac specific protein.2. Markers of live, bone and nervous system did not appear after Islet-1 induction account for the function of Islet-1 in promoting stem cell's differentiation is cardiac-specific.