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Cell line with sgRNA, plasmid and IRF7 function deficiency as well as construction method and application of cell line

A technology for loss of function and method of construction, applied to cells modified by introducing foreign genetic material, genetically modified cells, DNA/RNA fragments, etc.

Pending Publication Date: 2022-03-01
岭南现代农业科学与技术广东省实验室肇庆分中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is little information on the application of this technique in chickens

Method used

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  • Cell line with sgRNA, plasmid and IRF7 function deficiency as well as construction method and application of cell line
  • Cell line with sgRNA, plasmid and IRF7 function deficiency as well as construction method and application of cell line
  • Cell line with sgRNA, plasmid and IRF7 function deficiency as well as construction method and application of cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Construction of the sgRNA plasmid pX459-sgIRF7 for knocking out the IRF7 gene

[0056] 1. Design and screen to obtain the following two sgRNAs:

[0057] sgRNA-F: GGTCGTCGTTGCACTTGGAG (SEQ ID NO: 1);

[0058] sgRNA-R: CTCCAAGTGCAACGACGACC (SEQ ID NO: 2).

[0059] 2. Add Bbsl restriction sites (italics) at both ends of the sgRNA

[0060] sgRNA-F: CACCgGGTCGTCGTTGCACTTGGAG

[0061] sgRNA-R: AAACCTCCAAGTGCAACGACGACC c

[0062] The above primers were sent to Bioengineering Company for synthesis.

[0063] 3. Phosphorylation modification and annealing of the primers, the specific operation is as follows:

[0064] Resuspend the sgRNA with distilled water to a final concentration of 100 μM, add phosphate groups and anneal as follows, the specific process and formula can refer to Table 1.

[0065] Table 1

[0066]

[0067]

[0068] 4. Digest the pX459 vector with Bbsl:

[0069] Take 1 μg of the pX459 plasmid and digest it with Bbsl-HFTM for 30 minutes at 3...

Embodiment 2

[0079] Example 2 Screening IRF7 Functional Deficiency Cell Lines

[0080] 1. Culture DF-1 cells in 30mm flasks and transfect with pX459-sgIRF7 plasmid within 24 hours. For transfection, Thermo Fisher Lipofectamine LTX DNA Transfection Reagents Transfection Kit was used, and the transfection was carried out strictly according to the instructions of the kit.

[0081] 2. After the transfected cells were cultured for 36 hours, add puromycin to the culture dish to make the final concentration 5 μg / ml, and insist on replacing it with the cell culture medium containing puromycin (5 μg / ml) every 3 days. (If the cell density is too high, the cells need to be diluted and cultivated to facilitate the screening of a single cell line.) After 10 days, change the cell culture medium without puromycin every 5 days until obvious cell colonies appear (about 2 -3 weeks). We selected 10 cell colonies for single colony cell culture, and named these cell lines DF1-dIRF7-A1, DF-dIRF7-A2, DF1-dIRF7...

Embodiment 3

[0090] Example 3 Identification of the biological function of the IRF7 mutant cell line (KO IRF7)

[0091] 1. Cell growth characteristics

[0092] figure 1Shows the cell phenotype of wild-type DF-1 (DF1-WT) and KO IRF7 cells cultured for 12-96h. The results showed no significant difference between the two. The cells grew well at 12-36h, and after 48h, the cells appeared senescent or even died of old age (vacuole) due to contact inhibition.

[0093] The viability of DF1-WT and KO IRF7 cells was determined by CCK method and the growth curves were drawn. Methods as below:

[0094] The cell suspension was inoculated in a 96-well plate, 100 μl / well, 6 wells were set at each time point, and cultured at 37° C., 5% CO2. Take out the cells every 12 hours and add 10 μl CCK-8 to each well, mix gently to avoid air bubbles, then return to the incubator and incubate for 2 hours, then measure the absorbance at 450 nm with a microplate reader. Calculate the mean and standard error of th...

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Abstract

The invention belongs to the technical field of biology, and provides sgRNA for specifically targeting an IRF7 gene, and the sequence of the sgRNA is as shown in SEQ ID NO. 1 and SEQ ID NO. 2. The sgRNA primer and a proper carrier structure can form a plasmid, and the plasmid is used for knocking out a corresponding gene to realize deletion of an IRF7 function. Meanwhile, the invention also provides a plasmid, an IRF7 function-deficient cell line, and a construction method and application of the IRF7 function-deficient cell line. The cell line disclosed by the invention has the advantage that the IFN-beta mRNA level, the IRF1 mRNA level, the VIPERIN mRNA level and the CMPK 2 mRNA level can be remarkably inhibited. Compared with the mode that CN201910352557.4 only inhibits the expression level of interferon beta, inhibition in other aspects is more remarkable.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and more specifically relates to a sgRNA for specifically targeting IRF7 gene, a plasmid, a cell line with IRF7 function loss and its construction method and application. Background technique [0002] Interferon regulatory factor 7 (interferon regulatory factor 7, IRF7) is a member of the interferon regulatory factor (interferon regulatory factor 7, IRF) family, because it can interact with Epstein-Barr virus nuclear antigen-1 (EBNA -1) The SRE-like element on the gene Qp promoter (BamHIQ promoter, Qp) was found to bind and repress its transcription, which is now considered to be the most important interferon regulator for inducing type I IFN. Human IRF7 was first cloned in 1997 and is a common downstream factor of TLR and RIG-I signaling pathways. All IRF7s contain a conserved N-terminal DNA-binding domain (DNA-binding domain, DBD) and an IRF-associated domain (IRF-associated domain, IAD). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/12C07K14/47C12N5/10C12Q1/02
CPCC12N15/113C12N15/85C07K14/4702C12N5/0656G01N33/5044C12N2310/20C12N2510/00C12N2800/106G01N2500/10C12N2503/02
Inventor 陈瑞爱杨洁向程威王婷黄梅杨泽坤
Owner 岭南现代农业科学与技术广东省实验室肇庆分中心
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