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Method for analyzing an integration site of lentiviral vector in CAR-T cells, and primers

A technology of lentiviral vector and analysis method, which is applied to the analysis of the integration site of lentiviral vector in CAR-T cells. The primer sequence field of this method achieves the effect of good specificity, stability and high sensitivity.

Pending Publication Date: 2019-04-02
WUHAN BIO RAID BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of how to more sensitively, specifically and stably obtain the specific site where the exogenous fragment in the CAR-T cell is inserted into the genome

Method used

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  • Method for analyzing an integration site of lentiviral vector in CAR-T cells, and primers
  • Method for analyzing an integration site of lentiviral vector in CAR-T cells, and primers
  • Method for analyzing an integration site of lentiviral vector in CAR-T cells, and primers

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Embodiment 1

[0033] Example 1 Analysis of integration sites of lentiviral vectors in CAR-T cells

[0034] The primer sequences are shown in the table below:

[0035]

[0036] 1 Experimental method

[0037] 1.1 Extraction of total genome DNA of anti-BCMACAR-T cells

[0038] (1) Sample homogenate

[0039] Add 1ml DNAZOL Reagent to the CAR-T cell sample, and pipette 50 times, taking care to avoid air bubbles.

[0040] (2) DNA precipitation

[0041] Add 0.5ml of 100% ethanol to the above cell homogenate, turn it upside down 10 times, and let it stand at room temperature for 1-3 minutes, flocculents can be seen. Centrifuge at 15000g for 5min. Remove the supernatant with a pipette.

[0042] (3) DNA washing

[0043] Add 1ml of 75% ethanol, invert up and down 10 times, and centrifuge at 12000g for 1min. Carefully remove the supernatant with a pipette. repeat. Open the cap of the EP tube for 3-5 minutes, and let it dry in the air.

[0044] (4) DNA dissolution

[0045] According to th...

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Abstract

The invention discloses a method for analyzing an integration site of a lentiviral vector in CAR-T cells, and primer sequences applied to the method. The method uses an LTR region-specific primer of the lentiviral vector for unidirectional PCR to capture an LTR region inserted into a genome, and then uses two sets of adaptor primers for nested PCR amplification to obtain integration site sequences. The method of the present invention does not rely on restriction enzymes, has high sensitivity, specificity and stability, and can be used for cloning of lentiviral vector integration sites in complex or micro-samples, especially clinical samples and pre-clinical model samples. The integrated site sequences captured by PCR means are subjected to positioning analysis by a bioinformatics method soas to evaluate safety of the integration of the lentiviral vector in the genome of host cells.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for analyzing an integration site of a lentiviral vector in CAR-T cells, and a primer sequence used in the method. Background technique [0002] Lentiviruses (HIV-1, HIV-2) are a class of retroviruses. Existing lentivirus vectors come from multiple species, including human immunodeficiency virus (human immunodeficiency virus, HIV), simian immunodeficiency virus ( simian immunodeficiency virus, SIV) and so on. It is based on the lentiviral genome, removes genes that are not necessary for its expression, and replaces them with therapeutic genes. Lentiviruses can be integrated into the chromosomes of host cells for long-term stable expression, and compared with other retroviruses (such as adenoviruses), lentiviruses can not only infect dividing cells, but also infect non-dividing cells, based on the above advantages , Lentiviral vector has been widely used in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686
Inventor 张同存顾潮江高阳
Owner WUHAN BIO RAID BIOTECH CO LTD
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