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Genetic modification method for regulating and controlling animal endogenous gene expression

An endogenous gene and animal technology, applied to cells modified by introducing foreign genetic material, biochemical equipment and methods, and microbial measurement/testing, etc., can solve problems such as functional loss, lethality, and unclearness

Active Publication Date: 2012-09-19
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This life-cycle inactivation often brings the following problems: 1) gene inactivation leads to embryonic lethality or postnatal lethality, making it impossible to evaluate its function in the later stages of growth and development; 2) it is impossible to judge the phenotype of target gene inactivation Is it a result of the inactivation of the gene at the time of observation or a consequence of the inactivation of the gene in the early stages of growth and development; 3) The loss of function caused by the long-term inactivation of the gene is compensated by other gene functions, covering up the function of the gene Performance
Although the Cre-LoxP system solves the tissue-specific problem of gene knockout, the loss of function caused by it is irreversible; the transcriptional activation system regulated by tetracycline cannot down-regulate the endogenous expression of the target gene, and is only suitable for overexpression; Although the inducible RNAi system can be adjusted in time and space, the specificity and cytotoxicity of RNAi itself bring certain uncertainties to the research. Much lower than the level of cells, the reason is not clear
It can be seen that none of these systems can completely solve the problems that arise

Method used

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  • Genetic modification method for regulating and controlling animal endogenous gene expression
  • Genetic modification method for regulating and controlling animal endogenous gene expression
  • Genetic modification method for regulating and controlling animal endogenous gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Construction of tTR-KRAB and RtTR-KRAB transgenic plasmids driven by CAG promoter

[0090] The construction process of pcDNA3-CAG promoter-tTR-KRAB expression plasmid is as follows:

[0091] 1) pcDNA3.1(-)-Hygromycin (purchased from Invitrogen) was double-enzymatically digested with BglII and NheI to remove the original CMV promoter, filled in and ligated, and then used as a vector after double-digested with XbaI and BamHI to prepare for ligation into CAG promoter;

[0092] 2) SpeI and BamHI double digestion of PLVCT-tTR-KRAB or PLVCT-RtTR-KRAB plasmid (purchased from addgene company) to obtain the CAG promoter fragment;

[0093] 3) Ligate the vector fragment in 1) with the CAG promoter in 2) to obtain the pcDNA3.1(-)-Hygromycin-CAG promoter vector, which is double-cut with BamHI and KpnI, and then combined with PCR (using PLVCT-tTR- KRAB or PLVCT-RtTR-KRAB plasmid as a template) tTR-KRAB or RtTR-KRAB (also known as tTR-KRAB / RtTR-KRAB) fragments obtained after digesti...

Embodiment 2

[0095] Preparation of tTR-KRAB and RtTR-KRAB transgenic mice driven by CAG promoter

[0096] After the pcDNA3-CAG promoter-tTR-KRAB / RtTR-KRAB plasmid was digested with BciVI and EcoRI, the fragments containing CAG promoter-tTR-KRAB-polyA and CAGpromoter-RtTR-KRAB-polyA were recovered by tapping rubber, and passed through fertilized egg cell male prokaryotic DNA Transgenic Founder mice were obtained by microinjection (for the preparation of Founder mice, see Gordon K, Ruddle FH.1986. Genetransfer into mouse embryos. Dev Biol (NY1985) 4: 1-36. Kupriyanov S, Zeh K, Baribault H.1998. Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines. Transgenic Res 7:223-226).

[0097] A total of 101 mice were born by microinjection of CAG promoter-tTR-KRAB-polyA fragment, and 20 transgenic positive mice were identified by PCR twice; 19 transgene-positive mice were identified by two PCR tests. PCR identification results such as image 3 shown.

Embodiment 3

[0099] Construction of Nmyc-TRE-EGFP-Knockin targeting vector

[0100] Use ET-Clone technology to obtain the target targeting vector through two-step homology arm extraction. The steps are as follows: first, four homologous small arms a, b, c and d (which are respectively located at the 5' end of the 5' arm of the targeting vector) are obtained by PCR from Nmyc BAC (purchased from Gene Service Company, UK) with a length of 200-400 bp. ' end, 3' end of the 3' arm, 3' end of the 5' arm and 5' end of the 3' arm). The primers used are listed in Table 1.

[0101] Table 1

[0102]

[0103] Then connect a and b into pBR322-MK in the correct direction (see Pentao Liu, Nancy A. Jenkins and Neal G. Copeland. A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations. Genome Res. 2003 13: 476-484) SalI and SpeI; between the SpeI and BamHI sites, after SpeI linearization at the junction of a and b, the two extract a 14.78kb DNA fragment from Nmyc BA...

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Abstract

The invention relates to a genetic modification method for regulating and controlling animal endogenous gene expression. Tetracycline response elements are inserted into proper positions of endogenous genes of animals to be regulated and controlled according to fixed points through the gene recombination technology; and regulation and control genes are transferred into animal genomes by a transgene method, transgene animals are obtained, and the reversible expression regulation and control on the endogenous genes is realized under the effect of inductors of tetracycline or other analogues. A mouse endogenous gene induction expression regulation and control model built according to the method can be widely applied to the fields of gene function study, disease animal model building and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to an inducible model for regulating endogenous gene expression, a method for establishing the model, and an application of the model. Background technique [0002] A major challenge in the post-genomic era is to elucidate the functions of the thousands of newly discovered genes. The elucidation of the functional studies of these genes is directly related to the study and understanding of related processes such as development and disease. An effective way to study gene function at present is: at the cellular level, by up-regulating (overexpressing) or down-regulating (RNAi or mutation inactivation) the expression of the gene to study its impact on cell function; at the level of the whole animal , often by establishing transgenic animals to up-regulate the expression of the gene, or by establishing mutation and gene knockout animal lines to inactivate the function of th...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/85C12N5/10C12Q1/68
Inventor 费俭孙瑞林毛积芳王铸钢
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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