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The role of transcription factor klf4 in induction of iPS cell has become the hot spot in biological investigation field. This transcription factor is of great importance in various biological process including cell proliferation regulation, differentiation and embryonic development.In order to explore the infulence of overexpression or underexpression of klf4 on the process of liver regeneration in rats, firstly, the primers carrying the restriction enzyme sites and those without the enzyme sites were designed and synthesized referring to klf4 mRNA sequence. Then, the full length of klf4 cDNA was inserted into the multiple cloning site of the expression vector pEGFP-N1 to construct the pEGFP-N1-klf4 recombininat vector using molecular cloning technique. Likewise, according klf4 mRNA sequence, two RNA interference vectors (pGenesil-1.0-k814 and pGenesil-1.0-k1082) targeting klf4 gene were constructed through using the online design tools from several websites like ruibo, Genesil, Ambion and QIAGEN and searching for the siRNA designing-related articles. At he same time, ORF sequence of klf4 were obtained by digesting the expression vector pEGFP-N1-klf4 using two enzymes, and then the resulting klf4 ORF fragment was cloned into the multiple clone site of the interference vectors pGenesil-1.0-k814 and pGenesil-1.0-k1082 to construct the corresponding test vectors pGenesil-1.0-k814-klf4 and pGenesil-1.0-k1082-klf4. After sequencing, klf4 was cloned into the interference vectors for establishing the corresponding test vectors pGenesil-1.0-k814-klf4 and pGenesil-1.0-k1082-klf4. Finally, the obtained three kinds of vectors were in sequence infected into three groups of experimental rats by the technique of hydrodynamics-based transgene. After, And the related indexes of the rat liver infected with klf4, such as the morality rate of rat, liver index and the structural alteration of liver tissue, were examined by fluorescence microscopy technology, biological statistical method and routine HE staining, immunohistochemistry etc.Sequencing and agarose gel electrophoresis confirmed that the construction of klf4 expression vector, interference vector and test vector were successful. it was found that the the in vivo dynamic expression of interested fragment is consistent with the role of target vector by the method of gene addition and RNA interference in this paper, illustrating that the interested DNA fragment was expressed successfully. After pEGFP-N1-klf4 infection, the rat death rate was no significantly changed, whereas the the liver index were increased at 120h, and microstructures of liver tissue was slightly changed. Compared with normal group, the rat morality rate in the group infected with pGenesil-1.0-k814,-k1082 was significantly higher than those in control group, the increased liver index at 120h but the reduction at 168h, and the slight change in microstructures of live tissue. This study successfully cloned klf4 gene, established its expression vector, interference vector and test vector. And it proved that klf4 is related to liver regeneration according to in vivo expriement. All together, the research experience and data accumulated in this study provide an important reference and guidance for the future study on the function of klf4 on liver regeneration.
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