Establishment of vitro transcription plasmids of 6 pig source key genes for maintaining self-renewal of stem cells

An in vitro transcription and key gene technology, applied in the field of genetic engineering, can solve the problems of low gene knockout efficiency, defective newborn piglets, and high abortion rate during pregnancy

Inactive Publication Date: 2011-03-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in this process, there are problems such as low gene knockout efficiency, high abortion rate during pregnancy, and defects in newborn piglets; in addition, the isolation of pig stem cells has not been successful so far, so according to the experimental method of the Yamanaka research group, the fibroblasts were transferred into A few key stem cell genes turn fibroblasts into pluripotent stem cells
At present, the multi-gene transfection efficiency of fibroblasts using transfection reagents such as liposomes is very low, resulting in different amounts of genes transferred. There is a problem of biosecurity in removing pigs

Method used

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  • Establishment of vitro transcription plasmids of 6 pig source key genes for maintaining self-renewal of stem cells
  • Establishment of vitro transcription plasmids of 6 pig source key genes for maintaining self-renewal of stem cells
  • Establishment of vitro transcription plasmids of 6 pig source key genes for maintaining self-renewal of stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of gene in vitro transcription vector

[0035] 1. Determination of the sequence: According to the conditions of transcribing the gene into highly translatable mRNA in vitro, we selected the T7 promoter to initiate transcription; in addition, based on the literature and years of experience, we designed a 14bp CT sequence to enhance the transcription efficiency; finally, we inserted the gene A sequence containing an ATG initiation site was designed at the restriction site of mRNA to initiate mRNA synthesis. The sequence is shown in Figure 2: Figure 2A .In vitro transcription vector map; Figure 2B .In vitro transcription vector sequence 2 primer design, overlap extension PCR and connection with T vector: According to the designed sequence, we designed 2 pairs of overlapping primers, in which P1 and P2 overlap by 10bp, each containing 35 bases. P3 and P4 overlap the products of P1 and P2 by 7 bases, each containing 30 bases. The annealing tempera...

Embodiment 2

[0036] Example 2 Acquisition of control gene EGFP and cDNA of 6 key genes that maintain stem cell self-renewal

[0037] 1 Sequence determination and primer design: Query EGFP and pig Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28 mRNA sequences in the NCBI database, and then design PCR primers to amplify their sequences. Oct4 (NM_001113060.1), Sox2 (NM_001123197.1), c-Myc (NM_001005154.1), Klf4 (NM_001031782.1), Nanog (NM_001129971.1) and Lin28 (NM_001123133.1). Primers are designed according to the sequence obtained from the query and the optimal principle of correct expression. Both ends of the primers have endonuclease sites (NcoI, EcoRI and NheI) for connecting the in vitro transcription vector.

[0038] 2RT-PCR and connection with T vector: 20-day-old embryos were flushed out of pregnant sows, ground in liquid nitrogen, followed by RNA extraction, and then reverse transcribed. Then, using the reverse transcribed cDNA as a template, 6 PCR amplification of the genes to obtain cDNA...

Embodiment 37

[0039] Example 37 Connection of Gene to In Vitro Transcription Vector

[0040]Digestion of 17 genes and in vitro transcription vectors: Oct4-T plasmid was digested with EcoRI and NheI, Sox2-T, c-Myc-T, Klf4-T, Nanog-T and Line8-T plasmid were digested with NcoI and NheI . Then electrophoresis, cutting the gel, and recycling. The in vitro transcription vector was also digested with corresponding enzymes. Then, the restriction fragments of the seven genes were ligated with the in vitro transcription vector, transformed, and identified by shaking bacteria.

[0041] Linearization of the in vitro transcription vector of 27 genes: The in vitro transcription plasmid of the correctly identified 7 genes was cut with NheI to make it linearized, so as to facilitate the in vitro transcription.

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Abstract

The invention relates to the establishment of vitro transcription plasmids of 6 pig source key genes for maintaining the self-renewal of stem cells, which is characterized by comprising the following steps: firstly designing a vitro transcription null vector Vitro-Trans PMD-18T of the genes; obtaining enhanced green fluorescent proteins (EGFP) and complementary Deoxyribose Nucleic Acid (cDNA) of the 6 pug source key genes for maintaining the self-renewal of the stem cells and carrying out the enzyme cutting connection according to the corresponding enzyme cutting sites; carrying out the vitro transcription of 7 vitro transcription plasmids by utilizing a vitro transcription kit to obtain the corresponding messenger Ribose Nucleic Acid (mRNA); transfecting Vitro-Trans-EGFP into an embryo of a pig to form fiber cells; and transfecting the mRNA from the vitro transcription of the 6 genes into the embryo of the pig to be converted into pluripotent stem cells of the pig after forming the fiber cells, wherein the mRNA from the vitro transcription of the 6 genes can be translated at high efficiency.

Description

Technical field: [0001] The present invention utilizes genetic engineering technology to construct an in vitro transcription vector and an in vitro transcription plasmid of six key genes capable of inducing the transformation of porcine embryonic fibroblasts into porcine pluripotent stem cells, and is used for inducing porcine fibroblasts into porcine pluripotent stem cells. The invention provides basic conditions for establishing human disease models, and the invention belongs to the field of genetic engineering. Background technique: [0002] Pigs are the most suitable animals for human organ transplantation and the establishment of human disease models, but most of the pig disease models use gene knockout technology to obtain gene knockout somatic cells and then use nuclear transfer technology to generate oocytes that can continue to develop. Gene knockout pigs were obtained. However, in this process, there are problems such as low gene knockout efficiency, high abortion...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/12C12N15/63C12N5/10
Inventor 欧阳红生唐小春逄大欣周杨朱建国赖良学陈月段新平张明军
Owner JILIN UNIV
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