Promoter pLRRK2 and application thereof

A promoter, paav-plrrk2- technology, applied in the field of neuroengineering, can solve the problem of not being able to meet the specificity of gene delivery, etc., and achieve the effects of high labeling efficiency, improved expression specificity, and strong infection ability

Active Publication Date: 2021-04-23
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of the central nervous system, these serotypes are far from meeting the current need for gene delivery specificity

Method used

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  • Promoter pLRRK2 and application thereof
  • Promoter pLRRK2 and application thereof
  • Promoter pLRRK2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of PUC57-pLRRK2 vector

[0037] The pLRRK2 promoter is a partial sequence of the human leucine-rich repeat kinase 2 (LRRK2) gene. The sequence selects a sequence of 2201bp near the transcription start site of the LRRK2 gene, and replaces the ATG sequence contained in this sequence After the TTG sequence is used as the final promoter sequence, the nucleotide sequence of the promoter pLRRK2 is shown in SEQ ID NO:1.

[0038] The promoter pLRRK2 is artificially synthesized and then cloned into the PUC-57 vector to obtain the PUC57-pLRRK2 vector.

Embodiment 2

[0039] Example 2 Construction of recombinant adeno-associated virus vector Paav-pLRRK2-eYFP

[0040] In this embodiment, since YFP is used as the reporter gene of the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector Paav-hSyn-eYFP containing the YFP gene is selected as the carrier backbone to connect the pLRRK2 promoter, and the steps are as follows:

[0041] like figure 1As shown, the Paav-hSyn-eYFP vector was treated with restriction endonucleases xbalI-HF and kpnI-HF, and the PUC57-pLRRK2 vector was treated with restriction endonucleases xbalI-HF and kpnI-HF at the same time, digested at 37°C for 3 hours , the enzyme digestion system is shown in Table 1 and Table 2. After recovering the enzyme digestion product, the ligation premix (2×ligation premix, TAKARA) was used for ligation at 16°C for 30 minutes. The system is shown in Table 3, and the successfully ligated Paav was obtained. - hSyn-eYFP vector.

[0042] Table 1 Paav-hSyn-...

Embodiment 3

[0049] Example 3 Preparation of recombinant adeno-associated virus AAVDJretro-pLRRK2-eYFP

[0050] In this example, the virus was prepared by three-plasmid co-transfection method. Before virus preparation, plasmids required for packaging viruses need to be extracted, including recombinant adeno-associated virus vector Paav-pLRRK2-eYFP, packaging plasmid AAVDJ-retro-RCB and helper plasmids. Specific steps are as follows:

[0051] Preparation of cells: spread 293T cells in a petri dish containing full medium (10% fetal calf serum, 1% double antibody), and culture in an incubator.

[0052] Prepare transfection reagent: pipette 5.25 mL of ultrapure water, 75 μg of packaging plasmid, 75 μg of recombinant plasmid, 75 μg of helper plasmid, and 800 μL of calcium chloride solution, and mix gently. Add an equal volume of 2×HBS to the aforementioned reagents, vortex and let stand for 30 min.

[0053] Transfected cells: Add 20 μL of chloroquine to each plate of cells, add transfection ...

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Abstract

The invention discloses a promoter pLRRK2 and application thereof. The nucleotide sequence of the promoter pLRRK2 is disclosed by SEQ ID NO: 1. The promoter pLRRK2 can be applied to recombinant adeno-associated viruses, so that a foreign gene can be efficiently expressed in the new cortex, and the promoter pLRRK2 has a wide application prospect in the fields of neural circuit tracing and gene therapy.

Description

technical field [0001] The invention belongs to the technical field of neural engineering, and in particular relates to a promoter pLRRK2 and its application. Background technique [0002] The neocortex is part of the mammalian central nervous system, which is involved in many important functions including sensation, perception, voluntary movement, learning, and cognition. The analysis of the neural circuits connecting the neocortex and various nuclei can help us better understand the high-level functions of the brain. Research on the neocortex can also help us understand the pathogenesis of neurological diseases related to the cortex, and then find its treatment. However, there are currently no viral tools for efficient labeling of the neocortex, hampering neocortex research. [0003] Adeno-associated virus is a non-enveloped, single-stranded DNA virus. Recombinant adeno-associated virus (rAAV) is a recombinant virus modified on the basis of wild-type AAV. Recombinant a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N15/65A61K49/00
CPCC12N9/12C12N15/86C12N15/65C12Y207/11001A61K49/0045A61K49/0097C12N2750/14143C12N2800/107C12N2830/008Y02A50/30
Inventor 林剑邦陈晔菲路中华杨立新朱钰媛徐冬冬李梦奇
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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