Cloning And Function Analysis Of Porcine Muscle-specific Gene α-actin Promoter

How to start expression of foreign genes efficiently in receptor tissue is the key to genetic engineering. The expression of foreign genes critically depends on the start of transcription. The expression of gene in higher animals has temporal and spatial features. Tissue-specific promoter is also called organ-specific promoter. Under the effect of this kind of promoter, gene expression is often limited to certain specific tissues or organs, and also shows some characteristics like developmental regulation. However, separation and utilization of tissue-specific promoter is very little up to now, and accompanies by low activity and low tissue-specific defects. Tissue-specific promoters which have been separated and utilized in foreign countries have largely been applied for patents, which has greatly restricted the applications like transgenic animal pharmacy, livestock improvement and targeted therapy in various diseases in our country. Pig skeletal muscle-specific gene promoter a-actin was chosen as a research object. Promoter region with strong regulatory capability and muscle tissue- specificity is obtained by cloning, isolation and functional verification. Dual promoter strategy is built in order to improve the activity of promoter and to maintain tissue- specificity. The foundation for the future screening of promoter which can be effectively used is built. The main results are as follows:1. Input the DNA sequence of a-actin of pig skeletal muscle-specific gene into BLASTn database of NCBI, using comparative genomics approach, and get the upstream regulatory sequences of this gene.2. Select the upstream regulatory sequences 2006bp of pig muscle specific gene a-actin promoter which contained partial first intron, and submitted to TESS, TFSEARCH, MethPrimer and other bioinformatics softwares to predict and analyze its core promoter regions, transcription factor binding sites, different transcription elements and the distribution of CpG islands. TATA box was found in this sequence which contains a lot of muscle-specific transcription factor binding elements, such as MyoD, MEF2, CarG and MyoG, and so on.3. According to the online prediction results of bioinformatics software, deletion primers were designed, and the pMD18-T recombinant plasmid which contained 2006bp of a-actin promoter was used as a template, and six 5'flanking deletion fragments and two 3'flanking deletion fragments were got. First of all, these fragments were inserted into pMD18-T vector followed by being inserted into the upstream cloning site of pGL3-basic vector of the firefly luciferase reporter gene, and the corresponding reporter gene expression vector was built. These vectors were transitently transfected into C2C12, PK, and myotube and the relative activity of firefly luciferase was analyzed using dual luciferase reporter system. The expression activity of different fragments in same cells and same fragments in different cells were measured respectively. The results show that the expression activity of these fragments was highest in myotube, and next in C2C12, and the lowest was in PK cells. Finally,268bp core domain, the relative expression of which was highest and still had muscle-specificity, was obtained.4. Primers were designed. Taking plRES2-AcGFP1 as a template, core section of promoter CMV 560bp, SV40 231bp were obtained through PCR.5. By adding restriction enzymatic sites, the promoter CMV, SV40 and pig skeletal muscle-specific genes a-actin promoter which maintains muscle-specificity and higher expression activity are both inserted into the upstream polyclonal sites of firefly luciferase reporter gene of pGL3-basic vector respectively. Four reporter gene expression vectors of dual promoters are built which contain constitutive CMV or SV40 and pig skeletal muscle-specific gene promoter a-actin. These four kinds of recombinate vectors were transiently transfected into C2C12, PK and myotube respectively using lipofectamine method. The activity of luciferase was detected using dual luciferase reporter system. The results showed that:(1) The activity of pig skeletal muscle-specific gene promoter a-actin could be improved significantly by CMV or SV40, in which the effect of CMV promoter was particularly evident. (2) The activity of these four recombinate vectors containing dual promoters was highest in myotube, followed by C2C12, PK by turns.6.Taking pDsRed-N1 as a template, RFP with enzymatic Bg1â…¡and Hindâ…¢was obtained through PCR,and then inserted into the downstream of promoter Q8 which has already been inserted into polyclonal sites of pGL3-basic vector.The newly recombinate vector was named Q8-R.Using it as a template,Q8-RFP with enzymatic sites Claâ… and EcoRâ… was obtained,and then inserted into lentivirus vector pLenti7.3/V5-GW/lacZ.