RESUMO
According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of Ava II was found to be specific to ZFX gene. The results show that the combination of PCR with Ava II digestion is a simple and sensitive way to identify the tufted deer sex.
Assuntos
Proteínas de Ligação a DNA/genética , Cervos/genética , Processos de Determinação Sexual , Fatores de Transcrição/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Humanos , Fatores de Transcrição Kruppel-Like , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de SequênciaRESUMO
To investigate the expression of MT1F gene in hepatocellular carcinoma tissue and the growth suppression effect of exogenous introduction of MT1F gene on liver cell line HepG2 and to explore the potential application of MT1F gene in gene therapy of tumor. Eukaryotic expression vector of pCMV-MT1F plasmid was introduced into HepG2 line which expressed no MT1F protein originally with lipofectamine transfection method. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous MT1F gene on HepG2 cell line. The MT1F mRNA and MT1F protein were also detected in 60 pairs of surgical specimens of hepatocellular carcinoma by in situ hybridization and immunohistochemistry. The transfected HepG2 cell line grew more slowly than control HepG2 as shown by cell growth curves, the soft agar colony formation rate (3.8 percent vs. 7.4 percent, p <.01) and the average growth rate of tumor in SCID mice (30.9 +/- 6.9 vs. 70.3 +/- 5.6, p <.01). The expression level of MT1F mRNA and protein significantly increased in paracancerous tissue, normal tissue than in cancer tissues (75 percent, 70 percent vs. 16.7 percent by ISH and 66.7 percent, 60 percent vs. 10 percent by IHC, p <.01). Exogenous MT1F gene shows the strong effect of growth inhibition on HepG2 cell line. In the liver cancer tissue, MT1F shows down-regulated expression that supports the inhibited function of MT1F in cancer growth and suggests MT1F may have an important role in gene therapy of hepatocellular carcinoma.
