RESUMEN
In order to construct the recombinant retrovirus vector of human ngn3 gene and its packaging cell line, we successfully amplified the open reading frame (ORF) of ngn3 gene from human fetal pancreatic tissue by RT-PCR. The PCR products of human ngn3 gene was subcloned into pMD18-T vectors and sequenced. Results showed that its sequence was fully consistent with the ngn3 gene published in GenBank(GenBank Accession No. BC126468). The correct fragment was digested by EcoR I and Hpa I from recombinant pMD18-T vector and inserted into the same restriction enzyme sites of retroviral vector pMSCV-neo. We got recombinant retrovirus vector pMSCV-ngn3, which was identified by double restriction enzyme digestion and then transfected into PT67 cells by lipofectamine 2000. We established the PT67-ngn3 packaging cell line by G418 selection, which was detected by RT-PCR and immunohistochemistry staining. The detection results showed that the Ngn3 expressed at the mRNA and protein level in the packaging cell line. RT-PCR detection and electronic microscope analysis showed that the recombinant retroviral vector pMSCV-ngn3 was packaged into infectious virus particles and released into the supernatant of the cells. These results demonstrated that a PT67-ngn3 packaging cell line was successfully established, and this could facilitate the study of differentiation of the human fetal pancreatic progenitor cells into insulin-producing cells by using the ngn3 gene.
Asunto(s)
Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Genética , Diferenciación Celular , Línea Celular , Clonación Molecular , Feto , Vectores Genéticos , Genética , Células Secretoras de Insulina , Biología Celular , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Genética , Sistemas de Lectura Abierta , Genética , Páncreas , Biología Celular , ARN Mensajero , Genética , Proteínas Recombinantes , Genética , Farmacología , Retroviridae , Genética , Metabolismo , Células Madre , Biología Celular , TransfecciónRESUMEN
In order to construct the recombinant retrovirus vector of bovine sox2 gene and obtain infectious retroviral particles, we successfully amplified the ORF (open reading frame) of bovine sox2 gene from the primodial genital ridges of bovine embryo by RT-PCR. The cDNA of ORF was subcloned to pMD18-T vectors and verified that its sequence was highly homologous to the GenBank counterpart (GenBank Accession No. NM-001105463) by sequencing. The correct fragment was digested by EcoR I/Bgl II from recombinant pMD18-T vector and inserted into the same restriction sites f retroviral vector pMSCVneo. We got recombinant retrovirus vector pMSCV-sox2 which was transfected into PT67 by lipofectamine 2000 with pMIG (including green fluorescence protein) as a control. Flow cytometry analysis showed that its transfected efficiency was 68.3%. Subsequently, we established the stable cell strain by G418 selection which could produce virus. Its viral titer was up to 8.16x10(7) CFU/mL. This greatly facilitates the further study of bovine induced pluripotent stem cells induced from bovine somatic cells by defined factors.