ABSTRACT
Objective To perform mouse pMSV-Slfn5-GFP gene recombinant expression plasmid construction and gene structure analysis.Methods Total RNA was extracted from mouse liver and turned into cDNA by reverse transcription.Mouse Slfn5 coding sequence (CDS) fragment was amplified by PCR and connected with the pGEM-T Easy vector.The connected product was transferred the E.coli DH5a.The positive clones were selected for extracting plasmid,which was identified by double enzyme of restriction endonuclease Hpa Ⅰ and Xho Ⅰ.Then correct plasmid identified by enzyme digestion was sequenced by Macrogen USA.Then correct plasmid by sequencing was connected with pMSV-GFP by HindⅢ and Xbo Ⅰ,which was named as pMSV-Slfn5-GF.UCSC (http://genome.ucsc.Edu/) was used to analyze mouse Slfn5 and its family genomic structure.Slfn5 protein structural domain was determined by NCBI.Results Slfn5 full-length gene sequence was cloned into the expression vector pMSV-GFP,the fragment size was about 2.65 kb by enzyme digestion identification.The conservatism of AAA_4 protein domain in Slfn5 protein family was determined by phylogeny.fr.Conclusion Mouse full-length gene pMSV-Slfn5-GFP expression vector is successfully constructed.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the effect of Schlafen 1 (Slfn1) on the migration of endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>Rat bone marrow derived EPCs were isolated and cultured. Ad-Slfn1, ShRNA-Slfn1, ShRNA-control and Ad-control were transfected into EPCs respectively. The mRNA expression of Slfn1 and Cyclin D1 was examined by reverse transcriptase-PCR, and their protein expression was detected by Western blot. The migration of EPCs was examined by a modified Boyden chamber assay.EPCs cell cycle was determined using flow cytometry analysis.</p><p><b>RESULTS</b>Forty-eight hours after ShRNA-Slfn1 transfection, the mRNA and protein expression of Slfn1 in EPCs was significantly down-regulated compared to ShRNA-control EPCs (P < 0.05). Transfection of Ad-Slfn1 reversed these changes.Overexpression of Slfn1 reduced the migration capacity of EPCs while the silencing of Slfn1 by shRNA-Slfn1 increased the migration capacity of EPCs.In addition, cell cycle was arrested at G1 phase in Slfn1 overexpression group while transfection of shRNA-Slfn1 reversed these responses.Interestingly, the mRNA and protein expression of Cyclin D1 was significantly up-regulated after shRNA-Slfn1 transfection compared to ShRNA-control group (all P < 0.05), but overexpression of Slfn1 reversed these results, suggesting Cyclin D1 was involved in regulating EPCs cell cycle via Slfn1 signaling.</p><p><b>CONCLUSIONS</b>Slfn1 could reduce the migration capacity of EPCs via Cyclin D1 pathway.</p>