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Transcription factor system as well as preparation method and application thereof

A technology of transcription factor and construction method, applied in the application field of Alzheimer's disease gene therapy, can solve the problems of difficult to achieve in vivo application, insufficient recombinant virus titer, unsatisfactory packaging cells, etc., and achieve spatial learning. The effect of improving memory ability, improving spatial learning and memory ability, and reducing adverse reactions

Inactive Publication Date: 2014-05-07
NANJING MEDICAL UNIV
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  • Abstract
  • Description
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Problems solved by technology

First, the titer of the recombinant virus was not high enough, both at 10 1 TU / ml~10 3 Between TU / ml, it is difficult to meet the needs of in vivo applications; secondly, due to the complex biological properties of HIV, it is very difficult to establish stable HIV vector packaging cells like the commonly used mouse retrovirus vectors, and the established packaging cells are all not ideal

Method used

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  • Transcription factor system as well as preparation method and application thereof
  • Transcription factor system as well as preparation method and application thereof

Examples

Experimental program
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Embodiment 1

[0028] Example 1: Construction of recombinant lentiviral expression vectors of three neuron regeneration-related transcription factors Ascl1, Brn2, Pax6 According to the synthesis of DNA fragments of target genes Ascl1, Brn2, Pax6: According to human Ascl1 (Genbank accession number: NM_008553), Brn2 (Genbank accession number: NM_008899), Pax6 (Genbank accession number: NM_008899) gene sequence, design the target gene primer, its sequence is as follows:

[0029] (1) Ascl1: 5’-caagagcgcagccttag-3’; 3’-gcaaaagtcagtgctgaacg-5’.

[0030] (2) Brn2: 5’-aataaggcaaaaggaaagcaact-3’; 3’-caaaacatcattacctgct-5’;

[0031] (3) Pax6: 5’-gccagcaacacacctagtca-3’; 3’-ggggaaatgagtcctgttga-5’.

[0032] A gene fragment containing the DNA sequence of the target genes of the transcription factors Ascl1, Brn2, Pax6 was purchased from the human cDNA library (Shanghai Yingji Biotechnology Co., Ltd.), and the target genes of Ascl1, Brn2, and Pax6 were amplified by PCR. PCR product (PCR kit purchased from Takara...

Embodiment 2

[0036] Example 2: Recombinant plasmid lentivirus packaging

[0037] 1. Virus packaging steps

[0038] The lentiviral expression vector was constructed according to the method introduced by Zhou et al. (Zhou Letal., 2010). The recombinant lentiviral plasmid pGC-L-GFP-Ascl1, pGC-L-GFP-Brn2, pGC-L-GFP-Pax6 and the lentiviral packaging plasmid pHelper1.0 (Invitrogen, cat.No. 11668-500) and pHelper2.0 (Invitrogen, cat. No. 11668-500) were co-transfected with 293T cells. After 72 hours, the supernatant was collected and the growth medium was added at the same time. After 12 hours, the supernatant was collected again to obtain three recombinant lentiviral particles LV-Ascl1, LV-Brn2, and LV-Pax6.

[0039] 2. Specific steps for virus harvesting and concentration

[0040] Centrifuge the supernatant obtained in step 1 at 4000g for 10 minutes at 4°C to remove cell debris, and filter the supernatant through a 0.45um filter membrane in a 40ml ultracentrifuge tube; add the crude virus extract sam...

Embodiment 3

[0043] Example 3: Application of three transcription factor recombinant lentiviral particles in Alzheimer's disease gene therapy

[0044] Select 40 adult male C57BL6 mice and randomly divide them into four groups, set as blank control group, Alzheimer's disease model group, high-dose virus treatment group, low-dose virus treatment group, model group mice and treatment group. Aβ1-42 (purchased from -) was stereotactically injected into both hippocampus of mice, and the blank control group was given sterile saline. Three days before Aβ1-42 was modeled, the three recombinant lentiviral particles LV-Ascl1, LV-Brn2, LV-Pax6 were mixed in a ratio of 1:1:1 (virus titer 1*10 8 TU / ml), 2ul of the mixed virus mixture was injected into the bilateral hippocampus of mice in the treatment group through a stereotactic injection device. The blank control group and the model group were given the same dose of sterile saline and lentiviral LV-GFP as controls. .

[0045] One week after the treatment ...

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Abstract

The invention provides a transcription factor system as well as a preparation method and an application thereof. The invention relates to recombinant lentiviral vectors of three neurogenesis-related transcription factors Ascl1, Brn2 and Pax6 as well as a construction method and an application thereof. In particular, Ascl1, Brn2 and Pax6 target genes are obtained by utilizing a PCR (polymerase chain reaction) method and pGC-LV-GFP is used as a recombinant vector and synthesized DNA fragments of the three target genes are inserted into the recombinant lentiviral vectors, thus constructing GFP (green fluorescent protein) reporter gene built-in recombinant lentivirus plasmids pGC-GFP-Ascl1, pGC-GFP-Brn2 and pGC-GFP-Pax6 respectively containing Ascl1, Brn2 and Pax6. After lentivirus packaging, the three recombinant lentivirus plasmids are used for respectively transfecting a 293T cell and three recombinant lentivirus particles LV-Ascl1, LV-Brn2 and LV-Pax6 are obtained after culture and are concentrated by ultracentrifugation, and the titers of the packaged viruses are determined. Therefore, the transcription factor system can be applied to gene therapy of Alzheimer diseases and provides a new safe and effective therapy path for patients.

Description

Technical field: [0001] The invention relates to the fields of medicine and genetic engineering. More specifically, the present invention relates to a recombinant lentiviral vector of three neuron regeneration-related transcription factors Ascl1, Brn2 and Pax6 and its construction, and also relates to a recombinant lentiviral vector of three neuron regeneration-related transcription factors Ascl1, Brn2 and Pax6 in Al Application in gene therapy of Zheimer's disease. Background technique: [0002] Alzheimer's disease (AD), also known as Alzheimer's disease, is one of the most concerned neurodegenerative diseases in the world. AD is a disease characterized by progressive high-level cognitive dysfunction and loss of memory function. Studies have shown that the incidence of AD increases with age. The incidence is about 0.5% for 65-year-olds and 8% for those over 85. As our country gradually enters an aging society, the aging trend will increase the number of patients with Alzheime...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/867C12N15/66A61K48/00A61P25/28
Inventor 赵鹏顾爱华陈品颜青邹鹏王松涛
Owner NANJING MEDICAL UNIV
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