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Conditional inducible expression AsCpf1 lentiviral vector, construction method thereof and application to pig small intestine epithelial cell line construction

A technology of lentiviral vector and induced expression, which is applied in the direction of gastrointestinal cells, virus/bacteriophage, retroRNA virus, etc., can solve the problems of limited application, and achieve the effect of simple operation and convenient use

Pending Publication Date: 2019-04-02
FOSHAN UNIVERSITY
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Problems solved by technology

[0006] CRISPR gene editing technology is currently the most widely used and mainstream gene research tool, which has greatly promoted the research and development of medicine and life sciences. However, the biggest bottleneck in the application of human medicine is the extremely serious consequences caused by its off-target effect , which greatly limits its application in human medicine, and finer and more precise regulation has become one of the most urgent problems to be solved in current research

Method used

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  • Conditional inducible expression AsCpf1 lentiviral vector, construction method thereof and application to pig small intestine epithelial cell line construction
  • Conditional inducible expression AsCpf1 lentiviral vector, construction method thereof and application to pig small intestine epithelial cell line construction
  • Conditional inducible expression AsCpf1 lentiviral vector, construction method thereof and application to pig small intestine epithelial cell line construction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A method for constructing a conditionally induced expression of AsCpf1 lentiviral vector, which comprises the steps of:

[0029] 1) Digest the pCW-Cas9 vector with Nhe1 and Bamh1 to obtain a linearized Tet-on-3G lentiviral vector fragment with a length of 7595bp, such as figure 1As shown in A;

[0030] 2) Utilize primer 1 (sequence shown in SEQ ID NO1) and primer 2 (sequence shown in SEQ ID NO2), use the pLVX-AsCpf1 plasmid as a template to amplify the AsCpf1 gene to obtain an AsCpf1 gene fragment with a length of 4026bp, as shown in figure 1 As shown in B;

[0031] 3) Ligating the AsCpf1 gene fragment obtained in step 2) with the Tet-on-3G lentiviral vector fragment in step 1) by ligase, that is, constructing a Tet-on-3G-pLVX-AsCpf1 lentiviral vector with a length of 11585bp;

[0032] 4) Preliminary detection of the product obtained in step 3) with Nhe1 and Bamh1 double enzyme digestion, the detection results are as follows figure 1 As shown in C, the constructed Te...

Embodiment 2

[0034] A method for constructing a porcine small intestinal epithelial cell line that conditionally induces AsCpf1 expression, comprising the steps of:

[0035] 1) The Tet-on-3G-pLVX-AsCpf1 virus solution is obtained by packaging the Tet-on-3G-pLVX-AsCpf1 lentiviral vector with lentivirus. The specific lentivirus packaging scheme is as follows:

[0036] a. 293FT cell plating: one day in advance at 10cm 2 Cultivate 293FT cells in a culture dish, culture in 10% 293FT cell culture medium, and infect when the cell contact reaches 70-80%;

[0037] b. When infecting, take a clean 1.5mL centrifuge tube and add 300μL of DMEM, lentiviral packaging plasmids pSPAX2, pMD2.G and shuttle plasmid (pLVX-Tet-on-3G-AsCpf1), the usage of each plasmid is 3.5 μg, 10.4μg and 10μg, add 60μL Qiagen transfection reagent, mix well with a pipette gun, and let stand at 15~25℃ for 10~15min;

[0038] c. Wash the cells twice with PBS, add 6 mL of 10% 293FT cell culture medium, and add the mixture dropwise...

Embodiment 3

[0046] Example 3: Detection of conditionally induced expression of AsCpf1

[0047] The porcine intestinal epithelial cells conditionally induced the expression of AsCpf1 constructed in the above Example 2 were added with Dox 24h and stopped with Dox for 12h, 24h, 48h and 72h, RNA was extracted respectively, cDNA was synthesized, and the expression of AsCpf1 was analyzed by RT-PCR .

[0048] 1. Extraction method of total RNA from porcine small intestinal epithelial cells (Qiagen micro-extraction kit)

[0049] 1) For trace samples, add 80 μL of newly prepared lysate (lysate: 10 μL of mercaptoethanol in 1 mL of buffer RLT, prepared before use);

[0050] 2) Add 80 μL of 70% ethanol, mix with a pipette tip, do not centrifuge;

[0051] 3) Transfer the sample to the nucleic acid purification column provided in the kit, assemble a 2mL collection tube, cover it gently, centrifuge at 8000g for 15s, discard the breakthrough solution, and put it back into the collection tube;

[0052] ...

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Abstract

The invention discloses a method for constructing a conditional inducible expression AsCpf1 lentiviral vector. The invention also discloses a Tet-on-3G-pLVX-AsCpf1 lentivirus obtained by packaging thelentiviral vector and an application of the lentivirus to pig small intestine epithelial cell line construction. Pig small intestine epithelial cells are a cell material that is widely used in the study of intestinal nutrients and immunity. The present invention utilizes an AsCpf1-expressed lentivirus system regulated by a Tet-on-3G system to screen out an inducible-expressed stable-transfectionpig small intestine epithelial cell line. When Dox is added for induction, cells start a large amount of AsCpf1 expression within 24 hours, and when no Dox is added for induction, AsCpf1 expression disappears; and AsCpf1 decreases significantly after the Dox adding is stopped for 12h, AsCpf1 expression level is extremely low after the Dox adding is stopped for 24h and 48h, and no expression can beobserved after 72h.

Description

technical field [0001] The invention relates to the field of genetic bioengineering, in particular to a CRISPR-AsCpf1 system and its application in establishing pig small intestinal epithelial cell lines. Background technique [0002] The CRISPR / Cas system was first discovered in archaea, and it is a defense system used by archaea to resist the invasion of foreign genetic material. It recognizes foreign DNA sequences through specific small RNA guides, cuts them off by expressing nucleases, and silences the expression of foreign genes. It is because of this precise gene targeting function that the CRISPR / Cas9 system has been developed as an efficient gene editing tool. [0003] Zhang Feng et al. (2015) reported the CRISPR / Cpf1 system in a study published in the journal Cell. Cpf1 belongs to the Class II Type V CRISPR system, which has different advantages from the Cas9 system, which can make gene editing easier and more precise. Compared with CRISPR / Cas9, CRISPR / Cpf1 has t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N5/10
CPCC12N5/0679C12N15/66C12N15/86C12N2740/15043C12N2510/00C12N2800/107
Inventor 白银山朱翠温婕妤罗雁飞潘淳烨王丙云
Owner FOSHAN UNIVERSITY
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