Molucular Cloning Of Adipose Tissue-speciifcity Promoters Of Pigs

The technology of genetically modified pigs has made rapid progress in the pastdecades. However, lack of candidate genes and regulatory elements for breedinglimits the technology applications. The study on identifying pig tissue-specificitypromoters is very rare but useful. In this study, adipose tissue-specificity promotersof pigs were cloned, which could lay foundation for further research on theirfunctions.Firstly, online software “Digital Differential Display”(DDD)was used forscreening genes dominantly expressed in pig adipose tissues (both back fat andabdominal fat).100ESTs were got which expression level in adipose tissue washigher at least10fold than in other tissues.45ESTs stand for genes with annotationand the others may stand for novel genes. The top eight fold-changed genes(LGALS12、ADIPOQ、FABP4、LPL、LIPE、CIDEC、SDR16C5and SMAF1) werechoosen for GeneOntology analysis. Seven genes (or products) were existing as cellcomponent involving in the differentiation of adipocyte and the anabolism of fat,while only ADIPOQ had no function annotation in pigs. The tissue expressionprofiles of the eight genes were analyzed with real-time PCR. The data showed thatall eight genes were highly expressed in adipose tissue and confirmed the DDDanalysis results. Moreover, it was found that LGALS12、ADIPOQ and FABP4expressed in adipose tissue specifically. Therefore, we hypothesized that theirpromoters were adipose tissue-specificity regulatory elements.In order to get the three genes promoters region sequences, the positive BACclones containing the genomic sequence of them were respectively screened outfrom porcine genomic library. One clone of LGALS12, two clones of ADIPOQ andtwo clones of FABP4were got. With chromosome walking sequencing strategy, thesequence segments (2kb-10kb) upstream the initiation codon of three genes weregot. Then online software NNPP and PLACE were used to predict their corepromoters and cis-acting regulatory elements on the core promoters. Seven kinds of cis-acting regulatory elements were abundantly existing on the core promoters.Finally,3kb and5kb genomic segments upstream the initiation codon of FABP4were cloned as pig adipose tissue-specificity promoter candidate elements. The corepromoter regions of three genes were cloned and1.6kb fragments containingADIPOQ core promoter region was clone into the constructed vector ofpDsRed-Express2-1, which was without promoter element but with a reporter geneof red fluorescent protein downstream of the cloning site. This work lay thefoundation of the adipose tissue-specificity assay.