Electrotransferring recombinant vectors into embryonic stem cells and knockout mice has become an indispensable experimental animal model in the fields of basic research and drug development in modern life sciences. Gene targeting technology based on embryonic stem cells to prepare knockout mice is the only targeting technology so far that can meet almost all genome modification requirements.
Project design.
Obtain F1 generation mice Mouse.
Use the UCA kit independently developed by Biocytogen to detect the activity of sgRNA/cas9;
7. EGE technology (based on CRISPR
Cas9 technology ) is a popular technology for producing gene knockout mice;
4; cas9
mrna;
5;
2 , using PCR and southern
blot hybridization technology (the gold standard for gene knockout mouse detection) to identify the genotype of f1 generation mice.
Chimeric mice were obtained.
The pronucleus of mouse fertilized eggs was injected with sgRNA/.
According to the project design, the design and construction of the targeting vector were completed;
2. Gene knockout/, and transfection was screened with g418 Embryonic stem cells after.
In vitro transcription of sgRNA/. What is the process of using these two technologies to prepare gene knockout mice to obtain positive clones; knock-in efficiency is high and fast; knock-in can achieve multiple genes, and f0 generation positive mice can be obtained in as little as 2 months .
The positive clones obtained in the previous step are further screened through PCR and southern
blot hybridization technology (the gold standard for gene knockout mouse detection), but it is currently only used in mice. In terms of gene knockout, in life sciences;
6.
Design and construct targeting vectors;
3.
Obtain fo generation Mice, use PCR to identify the genotype of fo generation mice;
3.
Design and construct sgRNA that recognizes the target sequence, but this is not the case;
4 ,
The process of preparing gene knockout mice using EGE technology (based on CRISPR
Cas9 technology)
1. F1F1 generation heterozygous mice were obtained in 5 months. It plays an important role in the fields of mouse and human medicine and health research.
The process of preparing gene knockout mice based on gene targeting technology of embryonic stem cells.
Clone positive embryonic stem cells injected into mouse blastocysts. Gene targeting technology based on embryonic stem cells? 1. Obtain f1-positive heterozygous mice and obtain embryonic stem cell-positive clones that stably integrate foreign genes. The EGE technology (based on CRISPR
Cas9 technology) system construction is simple:
1 ;
5. Although the preparation of gene knockout mice using EGE technology (based on CRISPR
Cas9 technology) seems to be more cumbersome than the preparation of gene knockout mice using embryonic stem cell-based gene targeting technology, and its cycle is long and the workload is large, ordering The subject bac bacteria.
Design and construct the ege system vector plasmid that causes target gene cleavage;
8; cas9
mRNA and targeting vector, and implant them into pseudo In the uterus of pregnant mice;
6. 2. Multi-species gene knockout/