Construction And Identification Of Lentiviral Vectors Expressing Cat Pluripotericy Genes

In this experiment, the target genes of cat Oct4, Sox2, Klf4and c-Myc were obtained byreverse transcription-polymerase chain reaction （RT-PCR）,and according to the publishedsequences of cat Oct4, Sox2, Klf4, c-Myc genes in NCBI. A pair of specified primers containingthe restriction sites and protective bases were designed and synthesized. The NotI and BamHIrestriction sites were imported into primers of Oct4and the Not I and Nsi I restriction sites alsowere imported into primers of Sox2, Klf4, c-Myc. the target genes of cat Oct4, Sox2, Klf4andc-Myc were connected to the T vector, digested with restriction endonuclease, and weredirectionally connected to the lentiviral vector LV5carrying green fluorescence protein (GFP) toconstruct the reconbinant lentiviral vectors LV-Oct4、LV-Sox2、LV-Klf4and LV-c-Myc. Theproduction was respectively transformed into competent bacteria. The gene sequences wereanalyzed after identified by positive clones screening and restrictive enzyme digestion.Lipofectamine2000was used to transfect293T cells for packing lentivirus and testing the titer oflentivirus. The results are as follows:（1）The sequence of cat Oct4, Sox2, Klf4, c-Myc genes were cloned correctly by RT-PCRreaction, about1083bp，966bp，1458bp，1326bp nucleotide sequence of the cat Oct4, Sox2, Klf4,c-Myc genes were amplified. Correct sequence of cat Oct4, Sox2, Klf4, c-Myc genes wereconnected to the T vector.,then four recombinant plasmids were verified by sequencing. Thesequence homology of cloned Oct4, Sox2, Klf4, c-Myc gene were100%respectively to thereference sequence in GenBank.（2）The double enzyme digestion and DNA sequencing analysis revealed that the lentivirusvectors were successfully constructed and packed including Oct4, Sox2, Klf4and c-Myc. Thesequence of gene was consistent with objective sequence. Recombinant plasmid of LV-Oct4、 LV-Sox2、LV-Klf4and LV-c-Myc transfected293T cells by using lipofectamine2000, greenfluorescence was observed after24-48hours. Under fluorescence microscope,293T cells whichhave good shape can be seen, and293cells expressed GFP.The resulet showed that therecombinant lentiviral expression vector plasmid containing cat Oct4, Sox2, Klf4, c-Myc genewere successfully constructed.（3）Four recombinant lentiviral plasmid LV-Oct4、LV-Sox2、LV-Klf4and LV-c-Myc weretransfected into293T packaging cells with lipofectamine2000. The titer of lentivirus reached over107TU/ml. and obtained the packaging cell lines stably producing lentiviral particles.