RESUMO
AIM: To construct a lentiviral vector carrying human nuclear distribution protein C (hNUDC)-specific shRNA (sh-NUDC-F) and knock down hNUDC expression in Dami cells by infection of the lentivirus. METHODS: After labeling of green fluorescent protein (GFP), sh-NUDC-F was cloned into lentiviral vector pRRL-cPPT-CMV-X-PRE-SIN, and the high-quality plasmid was transfected into 293T cells to produce lentiviral particles by the calcium phosphate method. After high-speed centrifugation, lentiviral particles were collected and determined for its titer. The high-titer lentiviral particles were then transduced into Dami cells. By detecting the expression of GFP in lentiviral vector using a microscope, the transduction efficiency was readily figured out. And hNUDC protein level was detected by Western blotting. RESULTS: hNUDC was totally knocked down by the efficient transduction of Dami cells with the lentivirus carrying sh-NUDC-F. CONCLUSION: Lentiviral vector containing sh-NUDC-F can be successfully packaged in 293T cells and then efficiently transduced into Dami cells to silence hNUDC gene.
Assuntos
Proteínas de Ciclo Celular/genética , Vetores Genéticos , Lentivirus/genética , Proteínas Nucleares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução GenéticaRESUMO
AIM: Used RFP gene to construct a RNA interference vector for convenience to obtain the good effective hairpins sequence. METHODS: NUDC and RFP genes were cloned into pDs vector separately, resulting in pDs-NUDC- RFP. as above, human U6 promoter and 9 hairpins sequence of NUDC were cloned into the pDs- NUDC-RFP vector separately.The RNA interfererence vectors target to 9 points of NUDC were constructed. Construct- ed recombinant vectors and then were identified by restrictive digestion and DNA sequencing.293T cells were transfected with the recombinant DNA samples by the liposome complex method, and then fluorescence photographs were taken. RESULTS: Enzyme digestion and DNA sequencing showed that the target gene and shRNA fragments were correctly inserted in pDs vector. fluorescence photographs showed that shNUDC-A is the best effective fragment. CONCLUSION: The NUDC gene targeted shRNA and its vector are successfully constructed.
Assuntos
Fusão Gênica Artificial/métodos , Proteínas de Ciclo Celular/genética , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Células HEK293 , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Mapeamento por Restrição , Proteína Vermelha FluorescenteRESUMO
G banding karyotype analysis of peripheral lymphocytes in 217 patients with azoospermia or severe oligospermia were performed and the Y-chromosome AZFc region from 7 cases with Y chromosome abnormality was amplified by polymerase chain reaction (PCR). Out of 187 cases with azoospermia, 77 patients or 41.18% had chromosome abnormalities, including number and structural aberrations, heteromorphic chromosomes (Y chromosome heteromorphisms and pericentric inversion of chromosome 9) and 46, XX sex reversal. Two novel abnormal karyotypes were found: 46, XY, t(6; 14)(p21; p13) and 46, XY, t(8; 12)(p21; q24). Out of 30 patients with severe oligospermia, 4 had chromosome abnormalities, including structural aberration and 46, XX sex reversal. Therefore, aberration of the sex chromosome causes the most serious spermatogenic failure and certain breakpoints in the autosomes may also affect spermatogenesis. The deletion of AZFc also affects spermatogenesis.
