SgRNA with duck hnRNPA3 gene knocked out, cell line and construction method and application of cell line

A construction method and cell line technology, applied in the field of sgRNA to knock out duck hnRNPA3 gene, to achieve the effect of improving the amplification level

Active Publication Date: 2021-12-03
SICHUAN AGRI 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 solve the problem in the prior art that there is no duck-derived gene knockout cell line suitable for duck Tembusu virus research, which is not conducive to in-depth research on duck Tembusu virus, and provides a knockout sgRNA of duck hnRNPA3 gene, cell line and its construction method and application

Method used

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  • SgRNA with duck hnRNPA3 gene knocked out, cell line and construction method and application of cell line
  • SgRNA with duck hnRNPA3 gene knocked out, cell line and construction method and application of cell line
  • SgRNA with duck hnRNPA3 gene knocked out, cell line and construction method and application of cell line

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Experimental program
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Embodiment 1

[0030] Plasmids, cells, viruses: lentiCRISPR-v2 plasmid was purchased from Addgene, number #83480; plasmid pCMV-VSV-G was purchased from Addgene, number #8454; plasmid psPAX2 was purchased from Addgene, number #12260; immortalized duck embryo fibroblasts ( DEF) was purchased from Cybekon (Shanghai) Biotechnology Co., Ltd.; DH5α competent cells were provided by the Poultry Disease Control Center of Sichuan Agricultural University College of Veterinary Medicine; HEK293T cells were purchased from Wuhan Punosai Life Technology Co., Ltd.; Su virus (TMUV) was provided by the Center for Poultry Diseases, College of Veterinary Medicine, Sichuan Agricultural University.

[0031] 1. Construction of expression vector lentiCRISPR-v2-Blast plasmid

[0032] Schematic diagram of sgRNA targeting duck hnRNPA3 gene and expression vector lentiCRISPR-v2-Blast plasmid figure 1 shown. Use BsmBI endonuclease to linearize the lentiCRISPR-v2 vector. The linearization system and procedure are as foll...

Embodiment 2

[0045] Validation of TMUV proliferation on hnRNPA3 knockout cell lines

[0046] Set up test group 9-1clone and control group DEF (IM), the test group inoculates the DEF of hnRNPA3 gene knockout prepared in embodiment 1 into 12 well plates, and each group is set to repeat three times; Duck embryonic fibroblasts (naive DEF) were seeded into 12-well plates, and each group was set up in triplicate. When the cell density of the test group and the control group reached 80-90%, they were infected with the same amount of TMUV. After 24 hours of infection, the supernatant was discarded, and the total RNA was extracted by Trizol method, and it was reverse transcribed into cDNA . Using fluorescent quantitative PCR (RT-qPCR), the expression of TMUV E gene relative to the internal reference gene β-actin in the test group and the control group was detected, and the sequences SEQ ID NO.7 and SEQ ID NO.8 were used as the internal reference gene β-actin primers; use SEQ ID NO.9 and SEQ ID NO...

Embodiment 3

[0051] Validation of TMUV viral RNA translation on hnRNPA3 knockout cell lines

[0052] The test group 9-1 clone and the control group DEF (IM) were set up. The test group inoculated the hnRNPA3 gene knockout DEF prepared in Example 1 into a 24-well plate, and each group was set to repeat four times; the control group inoculated an equal amount of naive DEF were seeded into 24-well plates, and each group was set up in four replicates. When the density reaches 80-90%, transfect the TMUV translation system 5'UTR-Rluc-3'UTR respectively. After 4 hours of transfection, discard the supernatant, collect cell samples, and detect the level of Rluc in the cells, which represents the TMUV genome Translation situation. The results show that if Figure 5 As shown, the knockout of hnRNPA3 gene can significantly promote the translation level of TMUV RNA in duck embryo fibroblasts.

[0053] In summary, the present invention uses the CRISPR / Cas9 system to knock out the duck hnRNPA3 gene fr...

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Abstract

The invention discloses sgRNA with duck hnRNPA3 gene knocked out. The sgRNA is located on a fifth exon of the duck hnRNPA3 gene, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 1; the nucleotide sequence of an upstream primer corresponding to the sgRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of a downstream primer corresponding to the sgRNA is as shown in SEQ ID NO.3. The invention also provides an expression vector containing the sgRNA, the expression vector is a CRISPR/Cas9 recombinant lentiviral vector, and the expression vector is named as lentiCRISPR-v2-Blast. The invention further provides a cell line with the duck hnRNPA3 gene knocked out as well as a construction method and application of the cell line. According to the sgRNA with the duck hnRNPA3 gene knocked out, the cell line and the construction method and application of the cell line, the duck hnRNPA3 gene knocked out duck-derived cell line with stable passage is obtained for the first time, and the hnRNPA3 gene knocked out cell line can become an important tool for duck-derived virus amplification and research, and is especially beneficial to research of the function of the hnRNPA3 gene participating in TMUV life cycle.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sgRNA for knocking out duck hnRNPA3 gene, a cell line, a construction method and application thereof. Background technique [0002] The CRISPR / Cas system is an acquired immune system found in bacteria and archaea, which can achieve targeted cutting of specific genes. Among them, the Streptococcus-based Type II CRISPR / Cas9 system has become a widely used gene editing system due to its simple component composition [functional unit: Cas9 protein and sgRNA (single-guide RNA)] and relatively high efficiency. When a double-stranded DNA break (doublestrand break, DSB) caused by the CRISPR / Cas9 system appears in the genome of an organism, the cell will use its own repair mechanism, homologous recombination (Homology directed repair, HDR) or non-homologous end joining (Non -homologous end joining (NHEJ) repairs the damaged double-stranded DNA. Since mutations may be introdu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/12C12N5/10C12N15/66C12Q1/70
CPCC12N15/113C12N15/86C07K14/465C12N5/0656C12N15/66C12Q1/70C12N2510/04C12N2310/20C12N2740/15043Y02A50/30
Inventor 陈舜胡涛吴震陈维琼汪铭书程安春
Owner SICHUAN AGRI UNIV
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