CD28 gene overexpression vector and application thereof

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.

Active Publication Date: 2015-12-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CRISPR / Cas system generates a double strand DNA break (doublestrandbreak, DSB) at the target site, and the cell can be repaired by non-homologous end joining (NHEJ), resulting in a frameshift mutation of the gene and loss of function

Method used

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  • CD28 gene overexpression vector and application thereof
  • CD28 gene overexpression vector and application thereof
  • CD28 gene overexpression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CD28

[0035] Construction of embodiment 1CD28 gene overexpression vector

[0036] The process of constructing the CD28 gene overexpression vector is as follows: using the genome of the target organism as a template, PCR amplifies the left and right homologous arms of the first intron of the ROSA26 gene, the CD28 gene expression frame, and the OCT4 gene promoter, and then the homologous left and right Arm, CD28 gene expression frame, Cre expression frame and Neo gene expression frame regulated by the OCT4 gene promoter containing LoxP site, homologous right arm and negative selection marker DTA are connected in sequence, and constructed on the eukaryotic expression vector, namely 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] The Yorkshire pig ear tissue with excellent production performance was taken, and a po...

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. Select a sequence with the highest score from the candidate target sites and name it 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 the oligonucleotides of complementary pairing are synthesized respectively. As shown in Table 1, the underlines are restriction sites.

[0049] Table 1 Oligonucleotide sequence

[0050]

[0051] Dilute the synthesized pair of oligonucleotide sequences to 100 μM, add 10×PCR buffer to 10 μL reaction system (Table 2), mix well, anneal, 94°C, 5min; 37°C, 10min; 4°C, 5min. The resulting annealed product was ligated with the pX330 backbone digested with BbsI to obtain the CRISPR / C...

Embodiment 3CD28

[0055] Preparation and Identification of Example 3CD28 Transgenic Cell Line

[0056] The Yorkshire pig skin fibroblast cell line of 50 gestational age was selected as male, and the cells were recovered, cultured and subcultured according to the "Refined Cell Biology Experiment Guide" (J.S. Boniface et al., Zhang Jingbo et al., 2007 , Science Press). When the cells grow confluent to about 80%, digest and collect the cells (about 1×10 6), add 2 μg of each of the vectors constructed in Examples 1 and 2 and 100 μL of Nucleofector reagent, mix well, add to the electric shock cup, and perform electric shock transfection with the A-024 procedure. Then inoculated into a 10cm petri dish at a volume ratio of 1:20, at 37.5°C, 5% CO 2 cultured in an incubator. After 48 hours, the complete medium containing 500 μg / mL G418 (10% FBS+DMEM) was replaced every 3 to 4 days, and the concentration of G418 increased to 800 μg / mL after 96 hours. The formation of colony spots can be seen on the 8...

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Abstract

The invention provides a CD28 gene overexpression vector and application thereof. A target biological genome serves as a template, left and right homologous arms of a first intron of an ROSA26 gene, a CD28 gene expression frame and an OCT4 gene promoter are amplified through PCR (polymerase chain reaction), and the left homologous arm, the CD28 gene expression frame, LoxP-locus-containing Cre expression frame and Neo gene expression frame both regulated by the OCT4 gene promoter, the right homologous arm and a negative selection marker DTA are connected sequentially and are constructed to an eukaryotic expression vector to obtain the overexpression vector. The overexpression vector and a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) 9 target vector containing a first intron of a specific target pig ROSA26 gene are transplanted into a fibroblast of a pig fetus, a positive cell serves as a donor cell, an oocyte serves as a recipient cell, and a cloned embryo is obtained according to a somatic cell nuclear transfer technology; the cloned embryo is transplanted into the uterus of a pig for gestation to obtain a transgenic pig with a CD28 gene which is integrated at a fixed point in the first intron of the ROSA26 gene, wherein a marker gene of the transgenic pig is knocked out automatically.

Description

technical field [0001] The invention relates to the field of animal genetic engineering and genetic modification, in particular to a CD28 gene overexpression vector and its application. Background technique [0002] Mammals and other vertebrates have gradually formed a strong immune protection barrier in the process of responding to the invasion of various pathogenic microorganisms from the outside world and eliminating foreign substances to maintain their own balance. Includes humoral and cellular immunity. T cells are at the heart of most immune responses. After recognizing a specific antigen, T cells are activated, proliferated, and differentiated to have effector or auxiliary functions. initial Activation of T cells requires two stimulatory signals from antigen presenting cells. The first is the specific antigen stimulation signal generated by the combination of TCR / CD3 and the specific MHC-antigen peptide complex on the surface of antigen presenting cells (APCs); t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66A01K67/027
Inventor 索勋李秋艳黄广平刘贤勇李志远李向清付怡静王一丁田秀玲索静霞胡丹丹徐邢宾沈良才
Owner CHINA AGRI UNIV
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