Method for establishing SAMHD1 gene knockout cell line

A construction method and gene knockout technology, applied in the field of molecular biology, can solve the problems of incompleteness and instability of the SAMHD1 method

Inactive Publication Date: 2015-03-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for stably knocking out the SAMHD1 gene from the genome in view of the instability and incompleteness of the current transient knoc...

Method used

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  • Method for establishing SAMHD1 gene knockout cell line
  • Method for establishing SAMHD1 gene knockout cell line
  • Method for establishing SAMHD1 gene knockout cell line

Examples

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Embodiment 1

[0023] The construction of the SAMHD1 gene knockout cell line and its detection comprise the following steps:

[0024] (1) Select the target for CDS1 of the SAMHD1 gene, and design 2 pairs of recognition sequences, namely L1 / R1 and L2 / R2 (such as figure 2 ); select the target for CDS2 of the SAMHD1 gene, and design two pairs of recognition sequences, which are L3 / R3 and L4 / R4 (such as figure 2 ); L1 / R1, L2 / R2, L3 / R3, L4 / R4 are specifically:

[0025] L1: gtgtagccatgcagcgag, R1: cgcaacggggacgcttgg;

[0026] L2: gccatgcagcgagccgat, R2: tcatcgcaacggggacgc;

[0027] L3: atgaaatcacaggcgcat, R3: tttcaaaacgagactcat;

[0028] L4: aaatcacaggcgcattac, R4: agattttcaaaacgagac;

[0029] (2) Concatenate the gene fragments of the target recognition unit module according to the sequences of L1 / R1, L2 / R2, L3 / R3, and L4 / R4, and clone them into the pCAG-T7-TALEN(Sangamo)-Destination plasmid ( Such as figure 1 shown), a total of 4 pairs of TALEN expression plasmids ...

Embodiment 2

[0053] This example compares the knockout efficiency and stability of the SAMHD1 gene between the method described in the present invention and the small RNA interference technology. The currently used SAMHD1 knockout method is small RNA interference technology based on shRNA. In addition, HIV-1 Vpx expression plasmid can be transfected into target cells to degrade SAMHD1 protein. We compared the efficiency and stability of the above two methods with the SAMHD1 knockout method based on TALEN technology.

[0054] Method 1: using the method described in the present invention to knock out the expression of SAMHD1 in 293T cells;

[0055] Method 2: Transfect the HIV-1 Vpx protein expression plasmid Vpx-VPL into 293T cells to degrade the expression of SAMHD1;

[0056] Method 3: Synthesize shSAMHD1 5'-TGGAAATCTGTATGACATG and shCtrl 5'-TCGGCGCAGTCTAATTATA according to the SAMHD1 shRNA sequence commonly used in the literature, and transfect 293T cells;

[0057] The 293T cells treated...

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Abstract

The invention discloses a method for establishing an SAMHD1 gene knockout cell line. The method comprises the following steps: totally designing four pairs of recognition sequences aiming at SAMHD1 genes CDS1 and CDS2; respectively connecting gene segments of a target recognition unit module in series according to the four pairs of recognition sequences, cloning the gene segments into pCAG-T7-TALEN (Sangamo)-Destination plasmid, and establishing four pairs of TALEN expression plasmid pairs; and respectively transfecting 293T cell with the four pairs of TALEN plasmid pairs, and screening a cell line with the SAMHD1 genes knocked out by using neomycin. According to the method, frameshift mutation can be caused at target sites of the genome SAMHD1, a target gene knockout mutant is formed, and the aim of stably knocking out the SAMHD1 genes from the genome is achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a method for knocking out the SAMHD1 gene from the genome, which can be used for the research on the function of SAMHD1 and the construction of a cell line with stable loss of SAMHD1 expression. Background technique [0002] Recently, Laguette N et al. discovered a host restriction factor of HIV-1, SAMHD1, in myeloid cells that are difficult for HIV-1 to infect. This molecule can hydrolyze dNTPs, thereby inhibiting the replication of HIV-1 in human myeloid cells. The full length of SAMHD1 is 67020bp, including 16 exons, and the protein consists of 626 amino acids. Goldstone et al determined its structure and catalytic activity. The crystal structure revealed that SAMHD1 is a dimer, and its molecular structure includes the N-terminal SAM domain, HD domain and C-terminal region. The HD domain is characterized by a histidine / aspartic acid residue doublet motif, which is high...

Claims

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Application Information

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IPC IPC(8): C12N15/85
Inventor 靳昌忠吴南屏
Owner ZHEJIANG UNIV
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