Preparation method of sgRNA and HaCaT cell model targeting human genome sequence at high-frequency integration site of high-risk HPV

A technology of integration sites and cell models, applied in the field of medical biology, can solve problems such as cell models without HPV gene integration

Pending Publication Date: 2020-09-15
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] So far, there is still no mature cell model for HPV gene integration, which is largely due to the technical difficulty of site-specific knock-in of large fragments of genes in vitro. Therefore, it is necessary to establish a safe and effective gene knock-in system for HPV The cell model of gene site-specific integration provides technical support

Method used

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  • Preparation method of sgRNA and HaCaT cell model targeting human genome sequence at high-frequency integration site of high-risk HPV
  • Preparation method of sgRNA and HaCaT cell model targeting human genome sequence at high-frequency integration site of high-risk HPV
  • Preparation method of sgRNA and HaCaT cell model targeting human genome sequence at high-frequency integration site of high-risk HPV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Construction of sgRNA targeting plasmid and sgRNA screening

[0068] 1. Construction of sgRNA expression vector

[0069] (1) Design and synthesize sgRNA primers

[0070] Select the HPV integration sites with the highest frequency reported in the literature, 13q22 and 8q24. Referring to the HPV18 integration site in the human cervical cancer cell line HeLa, download the 250bp sequence of the 8q24 site 128230508-128230757 region of the human genome from the UCSC (http: / / genome.ucsc.edu / ) website, and use the online software (https : / / zlab.bio / guide-design-resources) to design sgRNA, select the top three with the highest scores, and obtain the sgRNA sequence double-stranded DNA, as shown in Table 1.

[0071]

[0072] Connect CAACCG to the left end of the sense strand of the sgRNA double-stranded DNA sequence, and connect AAACNNNC to both ends of the antisense strand (NNN represents the sequence of the antisense strand of the sgRNA), so as to carry out Bpil d...

Embodiment 2

[0122]Example 2. Preparation of linear Donor fragments containing HPV genes

[0123] (1) The Donor plasmid of the HPV 18 gene and the Donor plasmid of the HPV16 gene were synthesized and constructed by Shanghai Heyuan Biotechnology Co., Ltd. (such as Figure 5-6 shown); wherein, the Donor plasmid containing the high-risk human papillomavirus HPV18 gene (according to the target site of the sgRNA in the 8q24 region, select the upstream and downstream 500bp gene sequences of the CRISPR-Cas9 cleavage site as the left arm of the Donor plasmid respectively 1 and right arm 1, which are connected with the HPV18 gene fragment and the tag gene by a seamless cloning method to construct a Donor plasmid containing the HPV18 gene) consisting of left arm1 (as shown in SEQ ID No.8), HPV18 gene fragment, The tag gene and right arm1 (as shown in SEQ ID No.9) are constructed downstream of the ad element of the Donor backbone plasmid;

[0124] The HPV18 gene fragment is shown in SEQ ID No.7, com...

Embodiment 3

[0136] Example 3 Preparation of HaCaT cell model with site-specific integration of high-risk HPV18 gene

[0137] (1) Co-transfection of sgRNA plasmid and linear Donor fragment

[0138] Use the sgRNA targeting plasmid corresponding to 8q24-sgRNA2 obtained in the example and the linear HPV18 gene Donor fragment obtained in Example 2 to transfect the cells, take the HaCaT cells grown in the logarithmic phase, digest and inoculate them in a 6-well plate, and wait until the degree of cell fusion reaches When transfection is about 80%, each well is transfected (1 μg sgRNA + 1 μg Donor). For specific steps, please refer to the instruction manual.

[0139] (2) Cell drug screening flow sorting

[0140] 24 hours after transfection, according to the expression of the tagged gene, observe the green fluorescence of the cells under a fluorescent microscope to determine the transfection efficiency, then replace the cell culture medium with a complete medium containing 50 μM puromycin, and r...

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Abstract

The invention relates to preparation of an sgRNA and HaCaT cell model targeting a human genome sequence at a high-frequency integration site of high-risk HPV. According to the invention, specific sgRNAs are respectively designed for integration sites of HPV16 in a 13q22 region and an 8q24 region, and sgRNA double-stranded DNA is obtained; the sgRNA double-stranded primers are connected with a CRISPR/Cas9 vector to obtain an sgRNA expression vector, and sgRNA targeting plasmids are obtained through screening; a linear Donor fragment containing a high-risk human papilloma virus HPV gene is synthesized by using cloning construction and PCR amplification respectively, and the linear Donor fragment is used for co-transfecting HaCaT cells with the sgRNA targeting plasmids to obtain an HPV gene fixed-point integrated HaCaT cell model. According to the invention, the cell model integrated by HPV16 and HPV18 at high-frequency sites of a human genome is established by using a CRISPR/Cas technology, so that growth and proliferation of HaCaT cells are promoted, and a new method is provided for mechanism research of high-risk HPV integration in cervical cancer pathogenesis.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to the preparation of an sgRNA targeting a human genome sequence at a high-frequency integration site of high-risk HPV and a HaCaT cell model. Background technique [0002] Cervical cancer is the third largest malignant tumor in women in the world, and its incidence rate ranks first among gynecological malignant tumors. Human papilloma virus (HPV) infection has been proven to be the pathogenic factor of cervical cancer, the vast majority of cervical cancer is caused by persistent infection of high-risk HPV, HPV16 and HPV18 are the most important two high-risk Type HPV. In the early stages of HPV infection, the viral genome is replicated as episomal DNA outside the chromosomes along with the replication of the host genome. With the progress of precancerous lesions, viral oncogenes gradually integrate with the host genome, and most lesions have viral gene integration when they p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/85C12N5/10
CPCC12N15/1131C12N15/85C12N5/0636C12N2310/20C12N2510/00
Inventor 汪辉马丁任慈丁文成李晓敏熊劲风
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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