Method of knocking out spinal muscular atrophy SMN genes and cell model

A spinal muscular atrophy and gene knockout technology, applied in the field of genetic engineering, can solve problems that have not been seen yet, achieve good stability and reliability, low cost, and simple screening methods

Active Publication Date: 2014-07-09
江苏雄鸣医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, the retrieval of relevant materials including Chinese patents shows that the use of ZFNs technol

Method used

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  • Method of knocking out spinal muscular atrophy SMN genes and cell model
  • Method of knocking out spinal muscular atrophy SMN genes and cell model
  • Method of knocking out spinal muscular atrophy SMN genes and cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Knockout of SMN gene

[0034] 1. Design and construction of SMN gene ZFN plasmid knockout

[0035] According to the principle of ZFN technology, the DNA targeting recognition site is designed. The site is designed at the first exon shared by SMN1.SMN2 transcripts. After a lot of repeated tests, the determined target sequence information is:

[0036] TCCGTGCTGTTCCGGCGC ggcac AGGCCAGGTGAGGTCGCA (SEQ ID NO. 1). The underlined part at both ends is the ZFN binding region, and the lowercase letter in the middle is the ZFN cutting region. The plasmid construction was completed and identified by Sigma, USA. The specific information of its ZFN plasmid is as follows: figure 1 shown.

[0037]The specific construction method of the artificial zinc finger nuclease expression vector, the expression vector backbone is pAVX (Sigma), the vector contains pUC oir to promote the replication of the plasmid in prokaryotic cells, and has a kanamycin resistance gene; at...

Embodiment 2

[0073] Example 2 SMN gene knockout cell line mRNA expression identification

[0074] For the identification of cells with effective SMN knockout, extract RNA. For the method, refer to the instruction manual of the Trizol kit. After the total RNA is extracted, it is digested with DNase without RNAase activity to remove traces of genomic DNA. The RNA content is measured by UV spectrophotometer and confirmed by electrophoresis. RNA quality; cDNA was synthesized according to the instructions of the reverse transcription kit from Tiangen Company. Different primers were designed for (C → T) in the seventh exon of SMN1 and SMN2, and RT-PCR was carried out using different transcripts of SMN1 and SMN2 as templates to preliminarily determine the deletion of SMN1 and SMN2 according to the semi-quantitative situation.

[0075] However, we found that base deletions and base insertions in exons did not affect the stability of RNA transcribed by SMN1 and SMN2. Based on this conclusion, we...

Embodiment 3

[0096] Example 3 Western blot detection of SMN protein expression

[0097] For the obtained two SMN1- / - SMN2+ / - genotype Hela cell lines HS-66 and HS-1-7, when they grow to 80-90% in a 60mm cell culture dish, trypsinize, lyse the cells, and extract Protein, use a microplate reader to measure the protein concentration, take 45ug of the sample and load it on SDS-PAGE electrophoresis, use 10% separating gel, 5% stacking gel, PVDF membrane wet transfer, transfer time 1h, rabbit anti-SMN Antibody monoclonal antibody incubation 1h, Goat -Anti-rabbit secondary antibody was incubated for 1 hour, detected by ECL fluorescence, developed and fixed, and photographed. as attached Figure 4 As shown, compared with normal HeLa cells, the amount of SMN protein expressed by the SMN gene knockout cells was significantly reduced.

[0098]

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Abstract

The invention relates to a method of knocking out spinal muscular atrophy SMN genes and a cell model. The cell model is constructed by adopting the following steps: (1) constructing and designing zinc finger nuclease plasmids for knocking out the SMN genes; (2) transferring the constructed ZFN plasmids for knocking out the SMN genes into mammalian cells by using a lipidosome method; (3) detecting the effectiveness of the ZFN gene knockout by using a genome PCR product of a ZFN peculiar knockout site and DNA sequence determination; (4) specific to the cells, the ZFN genes of which are effectively knocked out, carrying out monoclonal separation and identifying cell knockout types one by one; (5) carrying out mRNA level identification on gene knockout cell strains; (6) carrying out protein expression level identification on the gene knockout cell strains. The spinal muscular atrophy pathology cell model can be used for research of SMA pathogenesis; medicines can be screened by detecting the level of protein expression of SMN compounds in the cell model.

Description

[0001] technical field [0002] The invention relates to the field of genetic engineering, in particular to a method for knocking out the SMN gene of spinal muscular atrophy and a cell model. [0003] Background technique [0004] Spinal muscular atrophy (SMA) is an autosomal recessive genetic neuromuscular disease characterized by pathological degeneration of motor neurons in the anterior horn of the spinal cord, proximal limb and trunk muscle weakness and atrophy, and eventually respiratory failure and even death. The incidence rate of live births is 1 / 6000~1 / 10000, and the population carrier rate is 1 / 40~1 / 60. It ranks the second among fatal autosomal recessive genetic diseases, second only to cystic fibrosis. The occurrence of this disease brings great physical and mental pain to the patient, and also brings a heavy burden to the family. [0005] With the development of molecular biology and genetics, it is now found that the Survival Motor Neuron (SMN) gene is the m...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
Inventor 陈实姜东旭黎寒
Owner 江苏雄鸣医药科技有限公司
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