Promoter pCALM2 and application thereof

A promoter, paav-pcalm2- technology, applied to the promoter pCALM2 and its application fields, can solve the problems such as the inability to meet the specificity requirements, and achieve the effects of high labeling efficiency, improved expression specificity, and wide application prospects.

Pending Publication Date: 2021-04-30
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
View PDF6 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of the CNS, these serotypes are far from meeting the current needs for specificity in gene delivery and gene manipulation

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Promoter pCALM2 and application thereof
  • Promoter pCALM2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of pUC57-pCALM2 vector

[0037] The pCALM2 promoter is a partial sequence of the calmodulin 2 (CALM2) gene, and the sequence is selected from the partial sequence near the transcription initiation site of the CALM2 gene and the partial sequence of the first exon, totaling 3020 sequences. The nucleotide sequence of the promoter pCALM2 is shown in SEQ ID NO:1.

[0038] The promoter pCALM2 was artificially synthesized and then cloned into the pUC-57 vector to obtain the pUC57-pCALM2 vector.

Embodiment 2

[0039] Example 2 Construction of recombinant adeno-associated virus vector pAAV-pCALM2-eYFP

[0040] In this example, because eYFP is used as the reporter gene of the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the eYFP gene is selected as the vector backbone to connect the pCALM2 promoter, and the steps are as follows:

[0041] like figure 1As shown, the pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and KpnI-HF, while the pUC57-pCALM2 vector was treated with restriction enzymes MluI-HF and KpnI-HF, and digested at 37°C for 3 h , the enzyme digestion system is shown in Table 1 and Table 2, after the recovery of the enzyme digestion product, the linking premix (2×ligation premix, TAKARA) was used for ligation at 16 ° C for 30min, the system was shown in Table 3, and the successfully connected pAAV was obtained - hSyn-eYFP vector.

[0042] Table 1 pAAV-hSyn-eYFP vector digestion syst...

Embodiment 3

[0048] Example 3 Preparation of recombinant adeno-associated virus AAV-retro-pCALM2-eYFP

[0049] In this example, the three-plasmid co-transfection method was used to prepare the virus. Before virus preparation, the plasmids needed to package the virus need to be extracted, including the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP, the packaging plasmid AAV-retro-RCB and the helper plasmid. Specific steps are as follows:

[0050] Preparation of cells: 293T cells were plated in culture dishes containing complete medium (10% fetal bovine serum, 1% double antibody), and cultured in an incubator.

[0051] Prepare transfection reagent: Pipette 5.25mL of ultrapure water, 75μg of packaging plasmid, 75μg of recombinant plasmid, 75μg of helper plasmid and 800μL of calcium chloride solution, and mix gently. To the aforementioned reagents, an equal volume of 2×HBS was added, vortexed and left to stand for 30 min.

[0052] Transfected cells: Add 20 μL of chloroquine to ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a promoter pCALM2 and an application thereof. The nucleotide sequence of the promoter pCALM2 is as shown in SEQ ID NO: 1. The promoter pCALM2 can be applied to recombinant adeno-associated viruses, so that exogenous genes can be efficiently expressed in the forebrain, and the promoter pCALM2 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, in particular to a promoter pCALM2 and its application. Background technique [0002] The forebrain belongs to the highest part of the brain and is the most complex and important nerve center in mammals. It is mainly composed of two parts: the telencephalon and the diencephalon. The telencephalon mainly includes the entire cerebral cortex, limbic system and basal ganglia, and the diencephalon mainly includes the thalamus and hypothalamus. It is involved in the regulation of many important physiological functions including movement, sensation, perception, autonomic nervous system, endocrine, feeding, learning, memory, cognition and so on. The analysis of forebrain neural circuits and their functions can help us better understand the high-level functions of the brain. The study of the forebrain can also help us better understand the pathogenesis of neurological diseases, and then find...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N15/65A61K48/00A61K49/00A61P25/00
CPCC07K14/4728C12N15/86C12N15/65A61K48/0058A61K48/0008A61K49/0045A61K49/005A61P25/00C12N2750/14143C12N2800/107
Inventor 王静怡陈晔菲朱钰媛林剑邦张建情徐冬冬李梦奇柏艳阳路中华
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap