Study On Differential Expression Genes Of Caski Cell Lines Induced By Exposing To The Space Environment By Suppression Subtractive Hybridization

Objective: To isolate genes expressed differentially from strainssubclonal 48A9 Caski cell line by Exposing to the Space Environmentand to explain the molecular mechanisms of the changes at the gene level.Methods: Super SMART cDNA synthesis and suppressionsubtractive hybridization (SSH) were performed to isolate differentiallyexpressed cDNA fragments from strains subclonal 48A9 cell line. cDNAfrom the 48A9 cell line were used as "tester", the other from the normalCaski cell line as "driver". Subtractive products were directly insertedinto T/A cloning vector, and then transformed into host bacteria, to set upa subtractive eDNA library of specially or highly expressed genes in fstrains subclonal 48A9 cell line. Those clones were screened andidentified by reverse Northern dot blot technique which the probes werelabeled with ³²P. Positive clones were sequenced and compared withknown sequences in the public databases of GenBank/EMBL/DDBJusing NCBI BLAST for homology analysis. The unknown fragmentswere then submitted to GenBank.Results: mRNA were directly extracted and purified in good quality.Double strand cDNA were reverse transcripted integratedly, and then cutby Rsaâ… into even length short segments. Ligation was identified highlyeffective. After two hybridizations, a subtractive library of differentiallyexpressed genes in strains subelonal 48A9 cell line was constructedsuccessfully by SSH. Nested-PCR was specificial, which amplified thedifferentially expressed genes exponentially. Subtractive products wereinserted into the T/A clone vectors and then transformed into DH5αcompetent cells successfully, which dispersed differentially expressedgenes of the library into a single copy. After differential screening 9clones were confirmed to be positive differentially expressed genesfragments. Sequences were submitted to GenBank for homology analysisto determine what kind of protein they were encoding, and confirm whichfragments were new ESTs. Then 4 sequences were a part of known genes,and the rests were verified unknown fragments. Conclusions: SSH is an effective approach to isolate differentiallyexpressed genes. Space environment exposure can make significantbiological impact on uterine cervix cancer cell by differential expressionof some genes and understating the mechanisms of these alternations mayabundant astrobiology research content and pave the way to cancertreatment.