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Porcine somatic cell transgenically-cloned plasmid pEGFP-N1-shRNA (Porcine Enhanced Green Fluorescent Protein-N1-short hairpin Ribonucleic Acid) and structuring method thereof

A pegfp-n1-shrna and construction method technology, applied in the field of embryonic biology, can solve problems such as mutual interference, failure to reach, EGFP expression interference, etc., and achieve the effect of wide application and convenient cloning operation

Inactive Publication Date: 2011-09-07
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0007] It is generally believed that the multiple cloning site of pEGFP-N1 is mainly used for the fusion expression of the target gene and EGFP, but the shRNA expression cassette contains a promoter and a transcription termination signal, so putting the shRNA expression cassette into the multiple cloning site may affect the downstream EGFP The expression causes interference or mutual interference, which fails to achieve the purpose of using pEGFP-N1 as a reporter gene

Method used

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  • Porcine somatic cell transgenically-cloned plasmid pEGFP-N1-shRNA (Porcine Enhanced Green Fluorescent Protein-N1-short hairpin Ribonucleic Acid) and structuring method thereof
  • Porcine somatic cell transgenically-cloned plasmid pEGFP-N1-shRNA (Porcine Enhanced Green Fluorescent Protein-N1-short hairpin Ribonucleic Acid) and structuring method thereof
  • Porcine somatic cell transgenically-cloned plasmid pEGFP-N1-shRNA (Porcine Enhanced Green Fluorescent Protein-N1-short hairpin Ribonucleic Acid) and structuring method thereof

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Embodiment 1

[0059] 1. Transformation of existing pSUPER-P2, pSUPER-G1, pSUPER-mG1 plasmids targeting PRRSV genes

[0060] 1) Design of primers:

[0061] Upstream primer: 5'-CGATTTAGAGCTTGACGGG-3' (SEQ ID NO.1)

[0062] Downstream primer: 5'-ACCGCGGTGGAAGGCCCTTTCGCCCTATAGTGAGTCGT-3' (SEQ ID NO2)

[0063] 2) The PCR reaction system is as follows:

[0064] 2×Taq PCR MasterMix 12.5μL

[0065] Upstream primer 1.0 μL

[0066] Downstream primer 1.0μL

[0067] pSUPER-P2 1.0 μL

[0068] wxya 2 O make up to 25.0 μL

[0069] Set on the PCR instrument at 94°C for 5min, followed by 94°C for 35sec, 52°C for 35sec, and 72°C for 40sec, for a total of 33 cycles, and after the end of the cycle, 72°C for 5min; the PCR product was identified by agarose gel electrophoresis and the fragment obtained was consistent with the expectation ( image 3 ), the size is 363bp.

[0070] 3) The PCR product is connected into a T vector to obtain a recombinant T vector for cloning, and the reaction system is as fo...

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Abstract

The invention relates to a porcine somatic cell transgenically-cloned plasmid pEGFP-N1-shRNA (Porcine Enhanced Green Fluorescent Protein-N1-short hairpin Ribonucleic Acid) and a structuring method thereof, belonging to the technical field of embryo biology. The plasmid pEGFP-N1-shRNA provided by the invention is obtained by inserting a target PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) gene shRNA expression cassette into a restriction enzyme cutting site EcoO109I of a vector pEGFP-N1 to simultaneously express EGFP and shRNA, wherein the target PRRSV gene shRNA expression cassette has the nucleotide sequences shown as SEQ ID NO.8, SEQ IF NO.9 or SEQ ID NO.10 and is subjected to functional verification. The structured plasmid pEGFP-N1-shRNA in the invention can be used for producing genetically modified cells, embryos or animals expressing the EGFP and expressing shRNA of the target PRRSV gene, so that a technical route for production of novel disease-resistant genetically modified species is got through.

Description

technical field [0001] The invention belongs to the field of embryonic biotechnology, and relates to a porcine somatic cell transgenic cloning plasmid pEGFP-N1-shRNA and a construction method thereof. The obtained transgenic plasmid can be used for porcine reproductive and respiratory syndrome in a targeted manner at the level of cells, embryos or whole animals Virus (PRRSV) suppression research lays the foundation for the cloning and production of new transgenic disease-resistant varieties. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS), also known as "PRRS", is characterized by reproductive disorders in sows and respiratory symptoms in newborn piglets. highly anticipated. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a high degree of variability. After infecting pigs, it will reduce the body's innate immune response. The virus can persist in pigs and easily cause mixed infections of other pathogens, which increases the...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66
Inventor 芮荣罗碧平马汝钧姜平
Owner NANJING AGRICULTURAL UNIVERSITY
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