Cloning Of Two Full Length Novel Genes And Their Initial Function

Introduction: Hepatitis B X antigen is a trans-activator which may alter the expression of cellular genes important to the development of hepatocellularcarcinoma. To test this hypothesis, recombinant retroviruses encoding the X antigen or the bacterial chloramphenicol acetyltransferase gene product were constructed, and used to infect HepG2-cells. The differences in gene expression between HepG2×and HepG2Cat cells were then evaluated by suppression subtractive hybridization and PCR. The full length of two differentially expressed genes were cloned by using Marathon cDNA full length amplification kit.Methods: Whole cell RNA was extracted separately from HepG2X and HepG2CAT cells using the Qiagen Rneasy RNA kit. PCR-select cDNA subraction was carried out by reverse transcriptase (RT)-PCR starting with 2 μ g of ployA~+ RNA isolated from the HepG2X and HepG2CAT cell lines. Adapters were then ligated to a fraction of Rsa-1 digested cDNAs generated by RT/PCR. The cDNAs were subjected to two rounds of subtractive hybridization against the PCR products from the cells in which the comparison was being made. The resulting products, now enriched for differentially expressed genes, were PCR amplified using primers that matched the sequence of the adapters. Following agarose gel electrophoresis, the unique fragments were then eluted from the gels and cloned into pT7Blue(?)T vector, and the both strands individually analyzed by sequence analysis. The sequences obtained were then compared to those in Genebank using the FASTA command in...