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Synthesizing method and application for LV-HDAC1shRNA lentivirus of target silence HDAC1 gene

A technology of targeted silencing and synthetic methods, applied in the application field of epigenetic modification drugs, can solve the problems of high experimental cost, low specificity of HDAC inhibitors, high price of HDAC inhibitors, etc., and achieve high transfection efficiency. Effect

Inactive Publication Date: 2017-09-19
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many deficiencies: 1. Broad-spectrum HDAC inhibitors are not specific, and may be dose-dependent, which may damage brain cognitive ability; 2. Although specific HDAC1 inhibitors can improve intracellular HDAC1 levels, but After cell passage and proliferation, the intervention effect shows a diminishing effect; 3. HDAC inhibitors are expensive, and the cost of in vivo experiments is high

Method used

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  • Synthesizing method and application for LV-HDAC1shRNA lentivirus of target silence HDAC1 gene
  • Synthesizing method and application for LV-HDAC1shRNA lentivirus of target silence HDAC1 gene
  • Synthesizing method and application for LV-HDAC1shRNA lentivirus of target silence HDAC1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Synthesis and screening of LV-HDAC1 with stable targeted silencing ability shRNA lentivirus

[0048] (1) Retrieve the mRNA sequence of human HDAC1 (NM_004964.2) on GenBank, and send 3 specific ones to Gemma Gene shRNA fragments, respectively shRNA1, shRNA2, shRNA3 , the sequence information is shown in Table 1;

[0049]

[0050] (2) Press siRNA : Lipofectamin2000 (1:0.05) transfected HEK293 cells, placed in CO 2 Cultivate in an incubator for 72 hours, extract total RNA from cells; use real-time fluorescent PCR technology (see Table 2 for primers, and see figure 1 -A), Western blot detection screened out the one with the best interference HDAC1 efficiency shRNA Fragment ( figure 1 -B,C, C is the grayscale analysis result of B), namely shRNA3. Note: *: with shRNA1 group compared to P shRNA2 group compared to P <0.05.

[0051]

[0052] (3) put shRNA3 The sense strand and antisense strand were dissolved in annealing buffer, placed in a w...

Embodiment 2

[0065] Example 2: Lentivirus LV-HDAC1 shRNA Infect hUC-MSCs and screen for HDAC1 shRNA - MSC cell line

[0066] The lentivirus LV-HDAC1 prepared in Example 1 shRNA Infect hUC-MSCs, screen HDAC1 that can target silencing of HDAC1 gene, regulate intracellular acetylation level, and proliferate well shRNA - MSC stable strain, the specific steps are as follows:

[0067] (1) Divide hUC-MSCs cells into 3~5×10 4 ml -1 Density seeding in 12 wells of a 96-well plate with a volume of 90 μl, cultured in a cell incubator for 24 hours;

[0068] (2) Preparation of infection reagents:

[0069] Prepare polybrene (M): dilute polybrene with Complete Medium to a final concentration of 50 μg / ml;

[0070] Prepare polybrene (E): dilute polybrene with Eni.s to a final concentration of 50 μg / ml;

[0071] Dilute the virus to three titers using Eni.s: Group A 1×10 8 TU / ml, group B 1×10 7 TU / ml, group C 1×10 6 TU / ml.

[0072] (3) From left to right on the abscissa: Complete Medium, polybr...

Embodiment 3

[0083] Example 3: Compound Induction Medium Neural Differentiation Induces MSC and HDAC1 shRNA - MSC cells, to assess the effect of lentiviruses on the neural differentiation of MSCs

[0084] The present invention uses neural differentiation induction medium to induce MSC and HDAC1 shRNA -MSC cells, qRT-PCR, immunofluorescence to assess lentivirus LV-HDAC1 shRNA For the impact on hUC-MSCs, the specific steps are as follows:

[0085] (1) Adjust MSC and HDAC1 with DMEM-LG medium shRNA -MSC cells at a density of 2 x 10 5 ml -1 , constant temperature culture to 60% confluence, DA differentiation medium pre-induction for 2 days, DB differentiation medium induction for 1 day, DC differentiation medium maintenance induction for 1 day, complete neural differentiation induction, and the characteristics of the induction medium are:

[0086] The DA differentiation medium is composed of 40ml FBS and 10μg bFGF per 1000ml DMEM-LG medium, and the final concentration of bFGF reaches 10 ...

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Abstract

The invention discloses a synthesizing method for LV-HDAC1shRNA lentivirus of a target silence HDAC1 gene. The synthesizing method comprises the steps: firstly searching an mRNA sequence of human HDAC1 on GenBank and synthesizing a specific shRNA segment; using Lipofectamin 2000 to transfect siRNA into a HEK293 cell, putting in a CO2 culture box to be cultivated for 72h and extracting cell total RNA; using a real-time fluorescent PCR technology to screen shRNA having the highest optimal interference HDAC1 effeciency; finally utilizing mU6-MCS-Ubi-EGFP tool carrier vector, pHelper 1.0 carrier vector and pHelper 2.0 carrier vector to synthesize lentivirus LV-HDAC1shRNA. Meanwhile, the invention further discloses application of the LV-HDAC1shRNA lentivirus serving as epigenetic modification drug capable of regulating HDAC1 expression in cells in high efficiency. The synthesized lentivirus disclosed by the invention changes an acetylation level of hUC-MSCs cell, obviously promotes proliferation of the hUC-MSCs cell and improvement of neural differentiation potential at the same time and is more favorable for follow-up cell transplantation therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LV-HDAC1 targeting silencing HDAC1 gene shRNA The method for synthesizing the lentivirus, the present invention also relates to the application of the lentivirus as an epigenetic modification drug for efficiently regulating the expression of intracellular HDAC1. Background technique [0002] In recent years, stem cell replacement therapy has brought hope for the treatment of central nervous system injury diseases including traumatic brain injury (TBI). Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have a certain ability to proliferate and differentiate, and are seed cells with little ethical controversy and high clinical application value. However, in view of the limited proliferation and differentiation efficiency of stem cells in vitro and in vivo, there is an urgent need for safer and more effective induction and regulation methods to improve their neurogenic differe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/867A61K48/00A61K31/713A61P25/00
CPCA61K31/713C12N15/66C12N15/86C12N2740/15043
Inventor 马珊珊关方霞邢衢王欣欣黄团结孟楠许玲刘腾飞杨波
Owner ZHENGZHOU UNIV
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