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Transcriptional control DNA in vitro transcription

A technology of in vitro transcription and sequence, applied in the field of genetic engineering

Inactive Publication Date: 2012-11-14
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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  • Transcriptional control DNA in vitro transcription
  • Transcriptional control DNA in vitro transcription
  • Transcriptional control DNA in vitro transcription

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The construction of embodiment 1 gene in vitro transcription vector

[0037] 1 Determination of the sequence: According to the conditions for the gene to be transcribed 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 transcription efficiency; finally, we inserted the gene A sequence containing an ATG initiation site was designed at the enzyme cleavage site to initiate mRNA synthesis. See Figure 2 for the sequence: 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, of which P1 and P2 overlapped by 10bp, each containing 35 bases. P3 and P4 overlap with the products of P1 and P2 by 7 bases, each containing 30 bases. The annealing...

Embodiment 2

[0038] Example 2 The control gene EGFP and the acquisition of 6 key gene cDNAs for maintaining stem cell self-renewal

[0039] 1 Sequence determination and primer design: query EGFP and porcine 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 were 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 to the in vitro transcription vector.

[0040] 2RT-PCR and connection with T carrier: Embryos about 20 days old were washed out from pregnant sows, ground with liquid nitrogen, and then RNA was extracted, then reverse transcribed, and then reverse transcribed cDNA was used as a template for 6 PCR assays. The PC...

Embodiment 37

[0041] The connection of embodiment 37 genes and in vitro transcription carrier

[0042]Enzyme digestion of 17 genes and in vitro transcription vectors: Oct4-T plasmids were digested with EcoRI and NheI, Sox2-T, c-Myc-T, Klf4-T, Nanog-T and Line8-T plasmids were digested with NcoI and NheI . Then electrophoresis, gel cutting, and recovery. The in vitro transcription vector is also digested with corresponding enzymes at the same time. Then, the enzyme-digested fragments of the 7 genes were connected to the in vitro transcription vector, transformed, and shaken for identification.

[0043] Linearization of in vitro transcription vectors for 27 genes: The in vitro transcription plasmids of 7 correctly identified genes were digested with NheI to linearize them to facilitate 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 vitrotranscription 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 uses genetic engineering technology to construct in vitro transcription vectors and in vitro transcription plasmids of six key genes capable of inducing porcine embryonic fibroblasts to transform into porcine pluripotent stem cells, which are used to induce porcine fibroblasts to become porcine pluripotent stem cells, It 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 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 produce oocytes that can continue to develop, thereby A knockout pig was obtained. However, in this process, there are problems such as low gene knockout efficiency, high abortion rate during pregnancy, ...

Claims

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Application Information

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Patent Type & Authority Patents(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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