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System for mutating ROSA26 genes and application thereof

A gene and cell line technology, applied in the system field of mutant ROSA26 gene, can solve the problems of host gene destruction, restriction of breeding and application of genetically modified pigs, silencing of foreign genes, etc., to achieve the effect of speeding up and efficiency

Pending Publication Date: 2018-05-11
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are mainly the following problems in the use of random transgenes: (1) The location of the integration of foreign genes into the host genome is uncontrollable, which may cause damage to the host genes, especially when using transposon vectors and viral vectors, they tend to integrate into the host transcriptionally active region ; (2) The copy number of the exogenous gene in the host genome is uncertain, and the genotypes of the obtained genetically modified animals are inconsistent during the passage; (3) The random integration of the exogenous gene leads to its invariance in various tissues of the host and among individuals. Differences in expression and individual phenotypes; (4) Random insertion of foreign genes, especially when viral vectors are used, the promoters of foreign genes are easily methylated, resulting in silencing of foreign genes
These shortcomings seriously restrict the breeding and application of genetically modified pigs

Method used

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  • System for mutating ROSA26 genes and application thereof
  • System for mutating ROSA26 genes and application thereof
  • System for mutating ROSA26 genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Using CRISPR / Cas9 technology to knock out the ROSA26 gene

[0061] 1. Design the target sequence of sgRNA targeting ROSA26 gene

[0062] According to the porcine ROSA26 gene sequence published by GenBank, the target DNA sequences of eight sgRNAs (denoted as sgRNA1-8) targeting the ROSA26 gene in CRISPR / Cas9 technology were designed. The target DNA sequences of the eight sgRNAs are shown in Table 1.

[0063] Table 1 Target DNA sequence of sgRNA

[0064]

[0065] 2. Construction of CRISPR / Cas9-sgRNA vector targeting ROSA26 gene

[0066] Add the CACC adapter sequence to the 5' end of the forward sequence of the target DNA of the eight sgRNAs in step 1, and add the AAAC adapter sequence to the 5' end of the reverse complementary sequence to synthesize the forward and reverse complementary sequences of the target DNA of the sgRNA, Its sequence is shown in Table 2.

[0067] Add each sequence with ddH 2 O was dissolved to 100 μM, and 15 μl of the two sequence...

Embodiment 2

[0086] Example 2. Using the target sequence 2 of Example 1 to construct ROSA26 site-specific integration transgenic pigs with Sfat1

[0087] 1. Construction of recombinant vector

[0088] 1. Construction of pCRISPR / Cas9-ROSA-sg2 vector

[0089] Synthesize upstream and downstream oligonucleotides 5'-CACCGCTCCTTCTCGATTATGGGCGGG-3' and 5'-AAACCCCCGCCCATAATCGAGAAGGAGC-3' with ddH 2 O was dissolved to 100 μM, and 15 μl of the upstream and downstream complementary sequence solutions were mixed, placed at 95 °C for 5 min, and then cooled to 16 °C at a constant speed of 0.2 °C / s to obtain the target DNA double-stranded oligonucleotide of sgRNA2. The CRISPR / Cas9 vector (the carrier in the UCA CRISPR / Cas9 Rapid Construction and Activity Detection Kit of Biocytogen Biotechnology Co., Ltd.) was digested with BsmB I of NEB Company, and the obtained vector backbone was combined with the target DNA double-stranded oligo of sgRNA2. Nucleotides are connected, and the recombinant vector obtai...

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Abstract

The invention discloses a system for mutating ROSA26 genes and application thereof. The system for mutating the ROSA26 genes provided by the invention is a CRISPR / Cas9 system. The system includes an sgRNA targeting the ROSA26 genes. A target sequence of the sgRNA is the following sequence: A1), A2), or A3), wherein A1) refers to a nucleotide sequence of sequence 2; A2) refers to a DNA sequence derived from A1) and having 75% or above identity with the DNA sequence defined by A1); A3) refers to a DNA sequence derived from A1) and hybridized with the DNA sequence defined by A1) under stringent conditions. The system also comprises Cas9 and donor DNA containing a Sfat1 gene expression cassette. Experiments prove that the ROSA26 gene can be successfully knocked out by using the system, and theSfat1 gene can be integrated in a targeted manner. Finally, an animal expressing Sfat1 is successfully obtained.

Description

technical field [0001] The invention relates to a system for mutating ROSA26 gene and its application in the field of biotechnology. Background technique [0002] Polyunsaturated fatty acids (PUFAs) are a class of fatty acids that have been extensively studied and concerned. PUFAs mainly include two types of ω-6 and ω-3 PUFAs, and each type includes multiple fatty acids ranging from short carbon chain (18C) to long carbon chain (22C) and containing multiple unsaturated bonds. More and more studies have proved that PUFAs have a wide range of biological functions. They participate in the formation of cell membranes and serve as signaling molecules in many cell response processes. They are closely related to the occurrence of various human diseases. It is extremely important for other mammals to develop properly and maintain good health. ω-6PUFAs are of little significance to the human body as a whole, and their excessive intake is harmful to human health. ω-3PUFAs have been...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N5/10A01K67/027
CPCA01K67/0276A01K67/0278A01K2217/072A01K2217/075A01K2227/108A01K2267/02C07K14/47C12N15/113C12N15/907C12N2310/10
Inventor 郗永义林艳丽王友亮左波吴晓洁钟荣斌陈红星
Owner ACADEMY OF MILITARY MEDICAL SCI