Notch1 Silence In Mouse Embryonic Stem Cells Impacts Dishevelled-1 And C-Jun Terminal Kinase-3's Expression

As a kind of highly Undifferentiated cells, embryonic stem cells(ESCs) have developmental totipotency and can differentiat into all types of adult animal's tissues and organs. The value of ESCs research is that these self-renewal and pluripotent stem cells can be widely used in embryonic development, replacement therapies, gene therapies and drug discovery. So stem cell techiques for therapeutic use are the prospective tendency of medical science.Notch1 gene expresses in many cell tissues, such as central nervous system, abdaminal salivary gland, hematopoietic cells, etc. The gene encoding a single-pass transmembrane receptor protein has been involved in the regulation of the signaling pathway of all sorts of cells' differentiation and development and has an impact on many growth and development process. There is a "lateral inhibition" in ESCs: Notch1 receptor in the surface of ESCs, in combination with its ligand Detal in the surface of an adjacant cell, inhibits the transcription of its downstream nerve differentiation genes, such as Mush1, Neuro D, which are the members of bHLH (basic helix-loop-helix) gene family. Furthermore the active Notch1 receptor inhibits ESCs's differentiation into neural cells. So after Notch1's down regulation, ESCs can be released from lateral inhibition and be promoted to differentiate into neural cells.Therefore, the aim of this sudy is to construct eukaryotic expression vector of siRNA (small interfering RNA) specific for Notch1 as a molecular target tool to inhibit Notch1 mRNA in mouse ESCs, and discuss the function of mouse ESCs after Notch1 inhibition by detecting the mRNA expression of dishevelled (Dvl)-1 and c-junterminal kinase (JNK)-3.Methods:1. To construct the siRNA expression vector of Notch1: according to the gensome sequences of Notch1 gene retrieved from GenBank, Ambion, siRNA design tools on web,was used to design two pairs of double strands siRNA. The siRNA fragments were cloned into T expression vector. After being identified by sequencing , target gene template was digested with Cla I /BamH I .Then the digested fragments were subcloned into pSINsi-U6 expression vector and identified by digesting.2. The recombinant plasmids were transiently transfected into mouse ESCs for 48 hours by Lipofectamine 2000, meanwhile G418 was uesed to selected the positive cell clones. At last the mRNA expression of Notchl, Dvl-1 and JNK-3 were detected by Real-Time PCR.Results:1. The pSINsi-U6-1 and pSINsi-U6-2 recombinant plasmids identificated by the restriction enzymes and the sequence analysis completely coincided with the design.2. The recombinant plasmids were transiently transfected into mouse ESCs by Lipofectamine 2000, and G418 induced their expression for 24 hours. Detected by Real-Time PCR, the mRNA expression level of Notch1 decreased remarkably in the groups of pSINsi-U6-1 and pSINsi-U6-2, but the control groups have no effect on ESCs.3. The effect of RNAi of pSINsi-U6-1 was more powerful than pSINsi-U6-2.4. After transfection, the mRNA expression of Dvl-1 and JNK-3 both increased, detected by Real-Time PCR.Conclusions:1. The siRNA eukaryotic expression vector pSINsi-U6-1 and pSINsi-U6-2 against mousse Notch1 mRNA has been successfully constructed and the recombinant vector effectively inhibited the expression of Notch1 in mouse ESCs, furthermore the mRNA expression of both Dvl-1 and JNK-3 increased.2. It can be inferred from the change of Dvl-1 and JNK-3' mRNA expression that the function of differentiating mouse ESCs were protected after Notch1 inhibition.