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A promoter and molecular technology, applied in the field of porcine myostatin gene promoter and its application, to achieve the effect of valuable gene resources
Active Publication Date: 2012-10-10
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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Problems solved by technology
However, at this stage, most studies focus on the knockout of the myostatin gene to obtain transgenic pigs with higher meat producti
Method used
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Embodiment 1
[0037] Embodiment 1, cloning and verification of porcine myostatin gene promoter
[0038] 1. Experimental materials
[0039] Table 1 Experimental materials
[0040]
[0041]
[0042] 2. Instruments and equipment
[0043] Table 2 Instruments and equipment
[0044]
[0045]
[0046] 3. Experimental method
[0047] 1) Pig genomic DNA extraction
[0048] Take 0.1 g of isolated pig ear muscle tissue samples from piglets, wash them, and cut them into pieces.
[0049] Add 300μl DNA extraction buffer (Tris-Hcl 10mM, EDTA 10mM, SDS 2%, NaCl 300mM, pH8.0), 8μl proteinase K (10mg / ml) to each sample, and digest at 55°C for 6 hours.
[0050] Extract twice with phenol, centrifuge at 10,000rpm for 10 minutes to get the supernatant, and remove the protein and phenol in the lower layer.
[0051] Extract again with chloroform / isoamyl alcohol and phenol, and centrifuge at 10,000 rpm for 10 minutes to get the supernatant.
[0074] The plasmid identified as positive by PCR and enzyme digestion was sent for sequencing. The result was that the plasmid was obtained by inserting sequence 1 in the sequence table between the MluI and BglII restriction sites of pGL3-bai sc, and the plasmid was named pGL3-MSTN. Embodiment 2, functional identification of porcine myostatin gene promoter
[0075] 1. Detection of transcriptional activity of porcine myostatin gene promoter
[0076] A. Preparation and verification of mutation vector of porcine myostatin gene promoter reporter system
[0077] 1. Experimental materials
[0078] Table 5 Experimental materials
[0079]
[0080]
[0081] 2. Instruments and equipment
[0082] Table 6 Instruments and equipment
[0083]
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Abstract
A pig myostatin gene promoter and its applications are disclosed in the present invention. The DNA fragment provided in the present invention which is derived from pigs is anyone of the following DNA molecules 1)-4): 1) the DNA molecule of SEQ ID NO:2 shown in the Sequence Listing; 2) the DNA molecule of SEQ ID NO:3 shown in the Sequence Listing; 3) the DNA molecule which hybridizes to the DNA sequence in 1) or 2) under strict conditions and has promoter function; 4) the DNA molecule which has more than 90% homology with the DNA sequence in 1) or 2) and has promoter function. The experiments in the present invention prove that the promoter provided in the present invention can drive the expression of firefly luciferase reporter gene. The promoter can also be built into the reporter vector and then the vector is transfected into cultured swine and human cells, and the activity and efficiency of the promoter are identified by accurate quantitative methods through reporter gene test.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a porcine myostatin gene promoter and application thereof. Background technique [0002] In the past ten years, transgenic technology has been developed rapidly and widely used, especially the combination of gene targeting and somatic cell cloning technology, which has made the gene-specific modification of transgenic animals a reality. The key to this technology is to make the exogenous gene site-specific integration and stable expression on the genome of the transgenic animal, and the expression of the exogenous gene is closely related to the transcriptional activity of its promoter. Therefore, obtaining a promoter with stable expression activity will greatly facilitate the research of transgenic animals. However, the types and quantities of promoters currently used in transgenic animals are very limited, mainly derived from viral promoters, such as cytomegalovirus (CMV), simian v...
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