cd28 gene overexpression vector and its application
A gene overexpression and gene expression technology, applied in the field of CD28 gene overexpression vector, can solve the problems of gene frameshift mutation and loss of function, and achieve the effects of low cost, enhanced effector function, and shortened time.
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Embodiment 1
[0035] Example 1 Construction of CD28 gene overexpression vector
[0036] The process of constructing the CD28 gene overexpression vector is as follows: using the target organism’s genome as a template, PCR amplifies the left and right homology arms of the first intron of the ROSA26 gene, the CD28 gene expression box, and the OCT4 gene promoter, and then the homology left and right Arm, CD28 gene expression box, Cre expression box and Neo gene expression box regulated by OCT4 gene promoter containing LoxP site, homologous right arm and negative selection marker DTA are connected in sequence and constructed on eukaryotic expression vector to get CD28 gene overexpression vector.
[0037] In this example, pigs were used as the target organism to construct the porcine CD28 gene overexpression vector pCDOCNDR. The specific construction process is as follows:
[0038] Take the Yorkshire pig ear tissue with excellent production performance and establish a porcine fibroblast cell line by th...
Embodiment 2
[0047] Example 2 Construction of CRISPR / Cas9 targeting vector
[0048] According to the CRISPR target sequence design website (http: / / crispr.genome-engineering.org / ), the target site of the first intron of the pig ROSA26 gene was predicted and analyzed. A sequence with the highest score was selected from the candidate target sites and named target1. Its sequence and reverse complementary sequence are 5'-TCCAGTCCCAGACATAGCAT-3' (SEQ ID NO. 21) and 5'-ATGCTATGTCTGGGACTGGA-3' (SEQ ID NO. 22) respectively, and complementary paired oligonucleotides are synthesized respectively. As shown in Table 1, the underline is the restriction site.
[0049] Table 1 Oligonucleotide sequence
[0050]
[0051] Dilute the synthesized pair of oligonucleotide sequences to 100μM, add 10×PCRbuffer according to the 10μL reaction system (Table 2), mix well, and anneal, 94℃, 5min; 37℃, 10min; 4℃, 5min. Connect the resulting annealing product to the pX330 backbone cut with BbsI to obtain the CRISPR / Cas9 targe...
Embodiment 3
[0055] Example 3 Preparation and identification of CD28 transgenic cell line
[0056] The 50 gestational age Yorkshire pig skin fibroblast cell line with male sex was selected, and the cell recovery, culture and passage were referred to the "Experimental Guide for Fine Lined Cell Biology" (JS Boni Fesnon et al., Zhang Jingbo et al., 2007 Years, Science Press). After the cells grow to about 80% confluence, digest and collect the cells (about 1×10 6 A), add 2 μg of the vector constructed in Examples 1 and 2 and 100 μL of Nucleofector reagent to mix well, add to the electric shock cup, and use the A-024 program for electric shock transfection. Then according to the volume ratio of 1:20 inoculated into a 10cm petri dish, 37.5℃, 5% CO 2 Cultivate in an incubator. After 48 hours, the complete medium (10% FBS+DMEM) containing 500 μg / mL G418 was replaced, and the medium was changed every 3 to 4 days. After 96 hours, the G418 concentration increased to 800 μg / mL. The formation of clonin...
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