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CRISPR/Cas9 system for preparing LMNA gene mutation dilated cardiomyopathy cloned pig nuclear donor cell and application of CRISPR/Cas9 system

A technology for dilated cardiomyopathy and recombinant cells, applied in DNA/RNA fragments, cells modified by introducing foreign genetic material, genetic engineering, etc., can solve problems such as imminent development needs

Pending Publication Date: 2021-03-19
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of large animal models of DCM for in-depth research on related mechanisms, and the need for development is imminent

Method used

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  • CRISPR/Cas9 system for preparing LMNA gene mutation dilated cardiomyopathy cloned pig nuclear donor cell and application of CRISPR/Cas9 system
  • CRISPR/Cas9 system for preparing LMNA gene mutation dilated cardiomyopathy cloned pig nuclear donor cell and application of CRISPR/Cas9 system
  • CRISPR/Cas9 system for preparing LMNA gene mutation dilated cardiomyopathy cloned pig nuclear donor cell and application of CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, the preparation of plasmid

[0076]The plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 was prepared, as shown in SEQ ID NO:1. Plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9, referred to as plasmid pX330.

[0077] The plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO was prepared, as shown in SEQ ID NO:2. Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, referred to as plasmid pKG-GE3.

[0078] The plasmid pKG-U6gRNA was prepared, as shown in SEQ ID NO:3.

[0079] Plasmid pX330, plasmid pKG-GE3, and plasmid pKG-U6gRNA are all circular plasmids.

[0080] The schematic diagram of the structure of plasmid pX330 is shown in figure 1 . In SEQ ID NO: 1, the 440-725 nucleotides form the CMV enhancer, the 727-1208 nucleotides form the chickenβ-actin promoter, and the 1304-1324 nucleotides encode the SV40 nuclear localization signal (NLS ), the 1325-5449th nucleotide encodes the Cas9 protein, and the 5450-5497th nucleotide encodes the nucleoplasmin nuclear localization signal (NLS)...

Embodiment 2

[0084] Example 2, Screening of Targets for LMNA Gene Knockout

[0085] Pig LMNA gene information: encoding prelamin-A / C; located on chromosome 4; GeneID is 100126859, Susscrofa. The protein encoded by the porcine LMNA gene is shown in SEQ ID NO:4. In the genomic DNA, the porcine LMNA gene has 16 exons, and the region between the sixth exon and the eighth exon of the porcine LMNA gene is shown in SEQ ID NO:5. In SEQ ID NO:5, nucleotides 1-157 are exon 6, nucleotides 2374-2499 are exon 7, nucleotides 2768-2938 are exon 8 son.

[0086] 1. LMNA gene knockout preset target and adjacent genome sequence conservation analysis

[0087] 18 newborn Congjiang pigs, including 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and 8 males (named A, B, C, D , E, F, G, H).

[0088] Using the genomic DNA of 3 pigs (female, 1, 2, 3) as templates, the primer pair consisting of LMNA-E7gRNA-JDF1 and LMNA-E7gRNA-JDR1 or LMNA-E7gRNA-JDF2 and LMNA-E7gRNA-JDR2 The primer pair was used for PCR ampl...

Embodiment 3

[0135] Example 3. Preparation of LMNA gene-edited monoclonal cells by somatic cell cloning

[0136] Primary pig fibroblasts were prepared from newborn Congjiangxiang pigs (female, blood type AO).

[0137] 1. Co-transfection

[0138] Plasmid pKG-U6gRNA (LMNA-E7-g1) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.92 μg plasmid pKG-U6gRNA (LMNA-E7-g1): 1.08 μg plasmid pKG-GE3.

[0139] Co-transfection was performed by electric shock transfection, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).

[0140] 2. After completing step 1, culture with complete culture medium for 16-18 hours, and then replace with new complete culture medium for cultivation. The total incubation time was 48 hours.

[0141] 3. After completing step 2, use trypsin to digest and collect cells, then wash with complete culture ...

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Abstract

The invention discloses a CRISPR / Cas9 system for preparing an LMNA gene mutation dilated cardiomyopathy cloned pig nuclear donor cell and application of the CRISPR / Cas9 system. The invention providesa kit which comprises sgRNALMNA-E7-g1 or plasmid pKG-U6gRNA(LMNA-E7-g1).sgRNALMNA-E7-g1, a target sequence binding region of the sgRNALMNA-E7-g1 or plasmid pKG-U6gRNA(LMNA-E7-g1).sgRNALMNA-E7-g1 is shown as nucleotides from the first to the 20th sites in SEQ ID NO: 6. The plasmid pKG-U6gRNA(LMNA-E7-g1) is transcribed to obtain sgRNALMNA-E7-g1. The invention also claims a method for preparing a recombinant cell. The method comprises the following step: co-transfecting a pig cell with plasmid pKG-U6gRNA(LMNA-E7-g1) and plasmid pKG-GE3 to obtain the recombinant cell. The CRISPR / Cas9 system lays asolid foundation for obtaining a dilated cardiomyopathy pig model by a gene editing means, and has great application value for research and development of dilated cardiomyopathy drugs and revealing of pathogenesis of the disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR / Cas9 system for preparing LMNA gene-mutated dilated cardiomyopathy cloned pig nucleus donor cells and an application thereof. Background technique [0002] Dilated cardiomyopathy (DCM, dilated cardiomyopathy, also known as congestive cardiomyopathy) is a primary myocardial disease of unknown cause. Most of the patients with clinical manifestations are middle-aged. The onset is more slow, sometimes up to more than 10 years. Symptoms are left or right ventricle or bilateral ventricle enlargement, accompanied by ventricular systolic dysfunction, with or without congestive heart failure, ventricular or atrial arrhythmia, shortness of breath and edema are the most common. In addition, there may still be embolism in the brain, kidney, lung, etc., and the condition is progressively aggravated, and death can occur at any stage of the disease. A genetic cause has been ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/87C12N5/10A01K67/027
CPCC12N15/113C12N9/22C12N15/8778C07K14/78A01K67/0273C12N2310/20A01K2207/20A01K2227/108A01K2267/0375Y02A50/30
Inventor 牛冬汪滔陶裴裴曾为俊王磊程锐马翔赵泽英刘璐黄彩云
Owner NANJING KGENE GENETIC ENG CO LTD
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