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Method for knocking out two mir-505 alleles

A biallelic, gene technology, applied in DNA/RNA fragments, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as unclear specific methods

Inactive Publication Date: 2015-05-27
DONGHUA UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0005] In 2010, Verduci L et al found that miR-505 could regulate the proliferation and senescence / apoptosis of mouse embryonic fibroblasts by acting on its target ASF / SF2 (alternative splicing factor) [Verduci, L, Simili M, Rizzo M, Mercatanti A, Evangelista M et al.(2010) MicroRNA(miRNA)-mediated Interaction between Leukemia / Lymphoma-related Factor(LRF)and Alternative splicing factor / splicing factor 2(ASF / SF2) affects mouse embryonic fibroblast senescence and apoptosis.J Biol Chem,285:39551-39563], Karni R et al found that transfection of ASF / SF2 in many cells can activate part of the mTOR signaling pathway [Karni R, Hippo Y, Lowe SW, Krainer AR (2008) The splicing- factor oncoprotein SF2 / ASF activates mTORC1.Proc Natl Acad Sci USA 105(40):15323-15327], but the specific way ASF participates in the regulation of mTOR is unclear

Method used

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  • Method for knocking out two mir-505 alleles
  • Method for knocking out two mir-505 alleles
  • Method for knocking out two mir-505 alleles

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Experimental program
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Embodiment 1

[0039] (1) Construction of neo-EGFP-Xho I-Nru I vector (with EGFP targeting vector)

[0040] A. Obtaining of EGFP Fragment

[0041] Using the pEGFP-C1 vector as a donor, the EGFP fragment was obtained by enzyme digestion. First digest the pEGFP-C1 vector with Nhe I, and use Klenow enzyme to blunt the sticky end to become a blunt end. On this basis, use Bgl II to perform a single enzyme digestion to obtain a blunt end at one end and a blunt end at the other end. EGFP fragment with cohesive ends. After digestion products were subjected to 1% agarose gel electrophoresis, as figure 1 As shown, a small fragment of 750 bp was indeed excised from the pEGFP-C1 vector, which is the EGFP fragment required for the experiment, and then the EGFP fragment was obtained by the method of rubber tapping and recovery.

[0042] B. Construction of neo-EGFP vector

[0043] After the pIRES-neo2 vector is linearized by EcoR V and BamH I, a linearized vector with a blunt end and a cohesive end is...

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Abstract

The invention relates to a method for knocking out two mir-505 alleles. The method comprises the following steps: constructing two targeting vectors for mir-505 alleles, replacing and knocking one mir-505 allele out of a cell of a mammal by mediating one targeting vector by using a CRISPR / Cas system, obtaining the cell with one mir-505 allele being knocked out by adopting a G418 resistance screening method, knocking another mir-505 allele out of the cell of the mammal by mediating another green fluorescent targeting vector by using the CRISPR / Cas system based on the cells, and obtaining the cell with two mir-505 alleles being knocked out by adopting a fluorescence screening method. The method is helpful to constructing a gene knocked-out cell model and has good application prospect.

Description

technical field [0001] The invention belongs to the field of gene knockout, in particular to a method for knocking out mir-505 biallelic genes. Background technique [0002] In 1987, the Japanese research group discovered a tandem spaced repeat sequence near the alkaline phosphatase gene of E.coli K12 [Ishino Y, Shinagawa H, Makino K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene produce.J Bacteriol,1987,169(12):5429-5433], in subsequent studies, it was found that such spacer repeat sequences widely exist in the genomes of bacteria and archaea, in 2002 In 2010, it was officially named by scientists as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)[Jansen R, van Embden J D, Gaastra W, et al. Identification of a novel family of sequence repeats among prokaryotes.Omics: a journal of Intearative Biology, 2002, 6(1): 23-33], [Mojica F J, Ferrer C, Juez G,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/113
Inventor 周宇荀邓倩云常雪莹王斯佳肖君华李凯
Owner DONGHUA UNIV
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