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Inducible shRNA (short hairpin ribonucleic acid) lentiviral expression vector and construction method and application thereof

A technology of expression vector and lentivirus, which is applied in the field of functional genomics research and can solve problems such as difficult operation

Inactive Publication Date: 2013-08-21
傅开屏 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Using such a system usually requires screening multiple clonal cells to determine which cell lines have successfully transcribed, which is difficult to operate

Method used

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  • Inducible shRNA (short hairpin ribonucleic acid) lentiviral expression vector and construction method and application thereof
  • Inducible shRNA (short hairpin ribonucleic acid) lentiviral expression vector and construction method and application thereof
  • Inducible shRNA (short hairpin ribonucleic acid) lentiviral expression vector and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0067] The inducible shRNA lentiviral expression vector specifically inhibits the oncogene PIK3CA, which requires the following steps:

[0068] 1. Construction of inducible shRNA lentiviral expression vector:

[0069] The shRNA expression vector provided by the present invention can be obtained by multi-step subcloning and transforming the lentiviral vector pLKO (from Sigma) containing the NEO selection gene. First, the restriction enzyme xho 1 and not 1 Excision of the constitutive U6-starting polymerase III promoter from the lentiviral vector. The resulting vector backbone was purified and recovered by agarose gel electrophoresis and QIAquick gel extraction kit (Qiagen). The tetracycline (Tet)-inducible polymerase III promoter U6 / TRE was chemically synthesized (SEQ ID 2), and used as a template to amplify and introduce the tetracycline-responsive element TRE sequence and TATA box using polymerase chain reaction (PCR) technology Sequences and Restriction Enzymes xho 1...

Embodiment 2

[0090] The inducible shRNA lentiviral expression vector specifically inhibits the oncogene KRAS gene, which requires the following steps:

[0091] 1. Construction of inducible shRNA lentiviral expression vector:

[0092] The shRNA expression vector of the present invention can be obtained by multi-step subcloning and transforming the lentiviral vector pLKO (from Sigma) containing the PURO selection gene (SEQ ID 5). First, the restriction enzyme xho 1 and not 1 Excision of the constitutive U6-starting polymerase III promoter from the lentiviral vector. The resulting vector backbone was purified and recovered by agarose gel electrophoresis and QIAquick gel extraction kit (Qiagen). The tetracycline (Tet)-inducible polymerase III promoter H1 / TRE was chemically synthesized (SEQ ID 1), and used as a template to amplify and introduce the tetracycline response element TRE sequence, TATA box sequence and restriction endonuclease xho 1 and not 1 Restriction site. product by ...

Embodiment 3

[0112] Inducible shRNA lentiviral expression vector for high-throughput drug target screening

[0113] The process of generating lentivirus from the 14K shRNA pool is as follows:

[0114] 5 L of virus was aliquoted and frozen at -80°C in preparation for infection experiments. The process of the infection experiment was carried out under the following conditions.

[0115]In order to determine the volume of virus that can produce a multiplicity of infection of 0.3-0.5 for each cell line, the virus was divided into 6 samples of different volumes (0-400 μL), and the cells were infected by the titer method, and the post-infection The cells were cultured in puromycin-containing and puromycin-free environments. Infected cells were filtered with a 40 μm cell strainer (manufactured by BD Falcon) before large-scale infection.

[0116] The infection experiments were divided into four groups, and each group of experiments was first divided into 3.7×10 7 Each cell was resuspended in 24...

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Abstract

The invention provides an inducible shRNA (short hairpin ribonucleic acid) lentiviral expression vector and a construction method and application thereof. The inducible shRNA lentiviral expression vector comprises an shRNA expression frame and a tetracycline repressor protein expression frame, wherein the shRNA expression frame comprises a first promoter, a tetracycline response element being combined with tetracycline repressor protein, a TATA box and an shRNA coding sequence, and the tetracycline repressor protein expression frame comprises a second promoter, a tetracycline repressor protein expression gene, an internal ribosome entry site (IRES) DNA (deoxyribonucleic acid) sequence and a marker gene. Compared with an existing inducible shRNA expression vector, the inducible shRNA lentiviral expression vector has the advantages of being capable of rapidly establishing inducible shRNA expressing a silent gene in various cell strains by using a single vector, having no need of clone selection of cells, and being applicable to in vitro culture and in vivo study of cells.

Description

technical field [0001] The invention belongs to the field of functional genomics research, and relates to a lentiviral expression vector, in particular to an inducible shRNA lentiviral expression vector and its construction method and application. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. [0003] In recent years, RNAi research has made breakthroughs. Because RNAi technology can specifically knock out or shut down the expression of target genes, this technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. [0004] In order to successfully implement RNAi, effective siRNA must first be obtained. The core of RNAi requires siRNA to effectively bind and act on the corresponding mRNA. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C40B40/06A61K48/00A61P35/00
Inventor 朱平
Owner 傅开屏
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