CN-115232793-B - Gene editing system for constructing ALS model pig nuclear transfer donor cells with SOD1 gene mutation and application thereof

Abstract

The invention discloses a gene editing system for constructing ALS model pig nuclear transfer donor cells with SOD1 gene mutation and application thereof. The invention provides a method for preparing recombinant cells, which comprises the step of co-transfecting pig cells with SOD1- g RNA1 (the binding region of a target sequence is shown as nucleotide 3-22 in SEQ ID NO: 18), SOD1- g RNA6 (the binding region of the target sequence is shown as nucleotide 3-22 in SEQ ID NO: 19), SOD 1-mut-ss 160 (shown as SEQ ID NO: 20) and NCN protein (Cas 9 protein or fusion protein with Cas9 protein) to obtain recombinant cells. The invention adopts CRISPR/Cas9 technology and ssODN homologous recombination technology to edit the point mutation gene of SOD1 gene, simulate the natural pathogenesis genetic characteristics of ALS, obtain single cell clone of accurate point mutation of SOD1 gene, and lay a foundation for culturing ALS disease model pigs by somatic cell nuclear transfer animal cloning technology in later period.

Inventors

• NIU DONG
• ZHAO ZEYING
• DUAN XING
• LIU LU
• WANG TAO
• MA XIANG
• TAO PEIPEI
• LIU YU
• ZENG WEIJUN
• WANG LEI
• CHENG RUI
• HUANG CAIYUN

Assignees

• 南京启真基因工程有限公司

Dates

Publication Date

20260505

Application Date

20210521

Claims (4)

1. 1. A method for preparing recombinant cells comprises replacing DNA molecule shown in SEQ ID NO. 21 in chromosomal DNA of pig cells with DNA molecule shown in SEQ ID NO. 20 to obtain recombinant cells; The implementation method of replacing the DNA molecule shown in SEQ ID NO. 21 in the chromosome DNA of the pig cell with the DNA molecule shown in SEQ ID NO. 20 comprises the steps of co-transfecting SOD1-gRNA1, SOD1-gRNA6, SOD1-mutant-ss160 and NCN protein into the pig cell, wherein the SOD1-gRNA1 is sgRNA, the target sequence binding region of the SOD1-gRNA1 is shown as nucleotide 3-22 in SEQ ID NO. 18, the SOD1-gRNA6 is sgRNA, the target sequence binding region of the SOD1-gRNA6 is shown as nucleotide 3-22 in SEQ ID NO. 19, and the SOD1-mutant-ss160 is a single-stranded DNA molecule shown in SEQ ID NO. 20; the NCN protein is shown as SEQ ID NO. 3; the preparation method of the NCN protein comprises the following steps: (1) Introducing plasmid pKG-GE4 into escherichia coli BL21 (DE 3) to obtain recombinant bacteria; (2) Culturing the recombinant bacteria by adopting a liquid culture medium at 37 ℃, then adding IPTG and performing induction culture at 25 ℃, and then collecting the bacteria; (3) Crushing the collected thalli, and collecting a crude protein solution; (4) Purifying the His 6 -tagged fusion protein from the crude protein solution using affinity chromatography; (5) Cutting fusion protein with His 6 label by enterokinase with His 6 label, and removing protein with His 6 label by Ni-NTA resin to obtain purified NCN protein; the plasmid pKG-GE4 is shown as SEQ ID NO. 1.
2. 2. The method according to claim 1, wherein the proportion of the porcine cells, SOD1-gRNA1, SOD1-gRNA6, SOD1-mutant-ss160 and NCN protein is 10 ten thousand primary porcine fibroblasts, 0.8-1.2. Mu.g of SOD1-gRNA1, 0.8-1.2. Mu.g of SOD1-gRNA6, 1.8-2.2. Mu. gSOD1-mutant-ss160 and 3-5. Mu.g of NCN protein.
3. 3. A kit comprising SOD1-gRNA1, SOD1-gRNA6, SOD1-mutant-ss160 and NCN protein; SOD1-gRNA1 is SOD1-gRNA1 described in claim 1, SOD1-gRNA6 is SOD1-gRNA6 described in claim 1, SOD 1-variant-ss 160 is SOD 1-variant-ss 160 described in claim 1, NCN protein is NCN protein described in claim 1; the kit is used for preparing recombinant cells, (b) preparing amyotrophic lateral sclerosis model pigs, and (c) preparing amyotrophic lateral sclerosis cell models or amyotrophic lateral sclerosis tissue models or amyotrophic lateral sclerosis organ models.
4. Application of SOD1-gRNA1, SOD1-gRNA6, SOD1-mutant-ss160 and NCN protein in preparation of kit; SOD1-gRNA1 is SOD1-gRNA1 described in claim 1, SOD1-gRNA6 is SOD1-gRNA6 described in claim 1, SOD 1-variant-ss 160 is SOD 1-variant-ss 160 described in claim 1, NCN protein is NCN protein described in claim 1; the kit is used for preparing recombinant cells, (b) preparing amyotrophic lateral sclerosis model pigs, and (c) preparing amyotrophic lateral sclerosis cell models or amyotrophic lateral sclerosis tissue models or amyotrophic lateral sclerosis organ models.

Description

Gene editing system for constructing ALS model pig nuclear transfer donor cells with SOD1 gene mutation and application thereof Technical Field The invention belongs to the technical field of gene editing, and particularly relates to a gene editing system for constructing a amyotrophic lateral sclerosis model pig nuclear transfer donor cell with SOD1 gene mutation and application thereof. Background Amyotrophic lateral sclerosis, also known as amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS), is a major type of motor neuron disease (Motor Neuron Disease, MND), commonly known as "freezing person disease", characterized by progressive degeneration of motor nerve cells (neurons) in the brain and spinal cord. Motor neurons control the muscle activity of the human body during movement, speaking, swallowing and breathing, without nerve stimulation, the motor neurons are gradually atrophic and degenerated, the muscles are gradually weakened to paralysis, and the speaking, swallowing and breathing functions are reduced until the respiratory failure dies. The disorder does not violate the sensory nerves of the human body, and therefore does not affect the intelligence, memory or feel of the patient. The progression of the disease is generally rapid, with an average life span of 3-5 years from the onset of symptoms, but with large fluctuations due to individual heterogeneity. "gradually frozen people" is listed by the world health organization as one of 5 absolute diseases juxtaposed with AIDS, cancer, etc., the incidence rate is about three ten thousandths, belonging to the world rare diseases. The international association of the "gradually freezing people" determines that the 21 th month of the year is the "world gradually freezing people day" at the international patient's congress held in denmark in 2000, and various related activities for recognizing motor neuron diseases are held all over the day, so that people attach importance to and social care of patients suffering from the terrible diseases are expected to be brought about through the activities. At present, the pathophysiological mechanism of ALS is not completely clear, no accurate epidemiological report of the incidence rate of ALS is yet available in China, but genetic factors related to ALS have been widely accepted. More than about 90% of ALS cases are sporadic (sporadic ALS, SALS), the remainder are familial inheritance (FAMILIAL ALS, FALS), and more than 30 genes have been identified as being associated with FALS. Among the most common and most studied genes are ALS1 (SOD 1), ALS10 (TARDBP), ALS6 (FUS), FTDALS (C9 orf 72), etc., which are associated with certain specific clinical features of ALS including disease onset, location and survival. SOD1 (superoxide dismutase, superoxide dismutase 1) is the first gene associated with FALS found by the university of hemp-province medical institute (UMMS) in 1993, and approximately one fifth of FALS is associated with SOD1 gene mutation. Currently, more than 180 mutations have been found in the human SOD1 gene associated with ALS, almost all of which are autosomal dominant inheritance. Gene mutation alters the conformation of SOD1 protein, and accumulation of high-toxicity hydroxyl radicals causes mitochondrial dysfunction, RNA metabolic disturbance and DNA damage. At present, it is widely considered that the neurotoxicity of mutant SOD1 proteins is caused by abnormal aggregation and binding of mutant SOD1 proteins on the surface of organelles such as mitochondria, which affect the functions of the organelles, or abnormal accumulation of mutant proteins, but the neurotoxicity is not confirmed by experimental animal models. Research on the occurrence and development mechanism of ALS caused by SOD1 mutation and research on corresponding medicines are all needed to be carried out on the basis of an animal model, and the animal model which is commonly used at present is a mouse model, however, the mouse has huge differences from human in aspects of body type, organ size, physiology, pathology and the like, and can not truly simulate normal physiological and pathological states of human beings. Pigs are major meat-fed animals for a long time, have the size and physiological functions similar to those of human beings, are easy to breed and raise on a large scale, have lower requirements on ethical morals, animal protection and the like, and are ideal human disease model animals. Gene editing is a biotechnology that has been greatly developed in recent years, and includes editing technologies from homologous recombination-based gene editing to nuclease-based ZFN, TALEN, CRISPR/Cas9 and the like, wherein CRISPR/Cas9 technology is currently the most advanced gene editing technology. Currently, gene editing techniques are increasingly applied to the production of animal models. Homologous recombination (HDR) is the exchange of DNA sequence information by sequence homology, i.e., the repair templat