Abstract:

BACKGROUND: We have been studied that hepatocytes can be induced to differentiate into pancreatic B-cells through the introduction of a few factors of key islet development genes, such as pancreatic and duodenal homeobox-1 (Pdx-1), neurogenin 3 (Ngn3), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA).
OBJECTIVE: To construct the plasmids using the pcDNA3.1(+) frame to introduce the Pdx-1, MafA and Ngn3 into the mice fetal hepatocytes and to detect the expressions of these genes.
METHODS: We amplified the target genes of Pdx-1, MafA and Ngn3 using templates of the cDNAs or the genome and then cloned to the eukaryotic expression vector pcDNA3.1 (+) multiple cloning sites. The eukaryotic overexpression vectors of pcDNA3.1(+)-MafA ORF, pcDNA3.1(+) -Pdx-1 ORF and pcDNA3.1(+) -Ngn3 ORF were established by Lipofectamine2000 mediated liposome transfected with mouse fetal hepatocytes of BNL CL.2. The expressions of three target genes were tested through reverse-transcriptional polymerase chain reaction and indirect immunofluorescence.
RESULTS AND CONCLUSION: The target genes were cloned correctly. The expressions of Pdx-1, Ngn3 and MafA in the fetal hepatocytes were validated by the detection of reverse-transcriptional polymerase chain and immunofluorescence test. We successfully constructed the eukaryotic overexpressed plasmids of pcDNA3.1(+)-MafA ORF, pcDNA3.1(+) -Pdx-1 ORF and pcDNA 3.1 (+) -Ngn3 ORF which could express the genes of Pdx-1, Ngn3 and MafA in the fetal hepatocytes.