A method for constructing an aav vector specifically expressing Cre in the ca2 region of mouse hippocampus and its application

A mouse and vector technology applied in the field of genetic engineering

A mouse and vector technology applied in the field of genetic engineering

CN110904154BActive Publication Date: 2022-04-22SOUTHEAST UNIV
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  • A method for constructing an aav vector specifically expressing Cre in the ca2 region of mouse hippocampus and its application
  • A method for constructing an aav vector specifically expressing Cre in the ca2 region of mouse hippocampus and its application
  • A method for constructing an aav vector specifically expressing Cre in the ca2 region of mouse hippocampus and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Cloning of Map3k15 promoter fragment

[0035] (1) DNA extracted from mouse brain (Qiagen DNeasy Blood and Tissue Kit, Catalog No.69504) was used as a template to amplify fragments of different lengths at the 5'-end of Map3k15 (Takara PrimerSTAR HS DNA polymerase, Catalog No. R010A).

[0036] A 3639bp fragment at the 5'-end of Map3k15 was amplified, which also included a part of Exon1 369bp downstream of the start codon. Use primer pairs:

[0037]Map3kpromo-F2:aggttggctgcaaactcact;

[0038] Map3kpromo-R: atcgtagaaggcatcgagcac;

[0039] Simultaneously amplify the 2293bp Map3k15 5'-terminal promoter fragment in the same way, using the primer pair:

[0040] Map3kpromo-F: gctggaactagggaaggatttc;

[0041] Map3kpromo-R: atcgtagaaggcatcgagcac;

[0042] At the same time, the 499bp Map3k15 5'-terminal promoter fragment was amplified in the same way, and the primer pair was used:

[0043] Map3kpromo-F3:ggaggaagtcaggggagggaggaaa;

[0044] Map3kpromo-R3: gcggggctgg...

Embodiment 2

[0047] Embodiment 2: Determination of Map3k15 startup efficiency

[0048] The three Map3k15 5'-end amplified fragments were subcloned into pGL4.0 plasmid respectively, using a non-ligase-dependent multi-fragment one-step cloning technique (Vazyme, ClonExpress MultiS One Step Cloning Kit, C113-02) and the following primer pairs :

[0049] 3639bp fragment

[0050] Map3k15-F2-Pgl4.1-F2: cctgagctcgctagcctcgagAGGTTGGCTGCAAACTCACTATG

[0051] Map3k15-R-Pgl4.1-R: cagtaccggattgccaagcttATCGTAGAAGGCATCGAGCACG

[0052] 2219bp fragment

[0053] Map3k15-F-Pgl4.1-F: cctgagctcgctagcctcgagGCTGGAACTAGGGAAGGATTTC

[0054] Map3k15-R-Pgl4.1-R: cagtaccggattgccaagcttATCGTAGAAGGCATCGAGCACG

[0055] 499bp fragment

[0056] Map3k15-F3-Pgl4.1-F: cctgagctcgctagcctcgagGGAGGAAGTCAGGGAGGGAGGAAA

[0057] Map3k15-R3-Pgl4.1-R: cagtaccggattgccaagcttGCGGGGCTGGCGGCTTCGAA

[0058] The multiple cloning sites of pGL4.0 are XhoI (5'-end) and HindIII (3'-end).

[0059] (2) Plasmid-transfected cells:

[0060...

Embodiment 3

[0077] Embodiment 3: Construction of rAAV-Map3k15-Cre plasmid vector

[0078] Using the rAAV-EF1α-NLS-Cre-WPRE-pA (Wuhan Privy Brain Science and Technology Co., Ltd., #143) plasmid as the vector backbone, the 499bp fragment of Map3k15 was cloned into MluI and SalI two enzyme cutting sites. Use primer pairs:

[0079] 143-map-F3-F:gttcctgcggccgcacgcgtGGAGGAAGTCAGGGAGGGAGG

[0080] 143-map-F3-R:ttcttgggggccatgtcgacGCGGGGCTGGCGGCTTCG

[0081] get as figure 2 The indicated plasmid vector was sent to Wuhan Privy Science and Technology Co., Ltd. for virus packaging.

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Abstract

The invention provides a construction method and application of an AAV virus vector capable of specifically expressing Cre recombinase in the CA2 region of mouse hippocampus. The invention cuts out the partial promoter region of mouse hippocampus CA2 specific expression gene Map3k15 and connects it with Cre recombinase, and clones it into the pAAV vector without initial promoter. The AAV vector with the Map3k15 promoter linked to the Cre recombinase constructed by the present invention can effectively specifically overexpress the Cre recombinase in the CA2 region of the mouse hippocampus to achieve specific regulation of neuron activity in the CA2 region, and establishes a small gene that does not depend on the Cre transgene. The mouse CA2 neuron tracing and regulation system provides an effective tool for studying the function of the CA2 region, and provides a basis for the subsequent study of the molecular mechanism of CA2 function.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a construction method and application of an AAV vector specifically expressing CRE in the CA2 region of mouse hippocampus. Background technique [0002] The harm to society and family caused by neurological diseases such as cognitive impairment, autism and senile dementia is attracting more and more attention from society and individuals. At present, our understanding of the nervous system is still in the exploratory stage, and our understanding of the functions and neural circuits of various brain regions and different nerve cells in mammals is very limited. [0003] Many studies have known that the hippocampus is a brain region that has an important impact on cognition, learning, memory, and social interaction. In recent years, the CA2 region of the hippocampus has been confirmed to be related to social memory, but its molecular mechanism is still unclear. In order...

Claims

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Application Information

Patent Timeline
22 Apr 2022
Publication
CN110904154B
IPC
C12N15/864; C12N15/64; C12N15/65; C12N15/113
CPC
C12N15/86; C12N15/64; C12N15/65; C12N9/12; C12Y207/11025; C12N2750/14143; C12N2800/30; C12N2830/008
Inventors
李默怡; 彭思琦