CN114410683B - RIM3-RNAi based on Cre-lox recombination system and application thereof - Google Patents
RIM3-RNAi based on Cre-lox recombination system and application thereof Download PDFInfo
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Abstract
The invention is suitable for the technical field of biomedicine, and provides a RIM3-RNAi based on a Cre-lox recombination system, which comprises an RNAi expression vector of Cre enzyme-dependent rAAV expression, wherein the expression vector comprises the following components in sequence: AAV2ITR, promoter of excitatory neuronal expression CaMKII alpha, cre enzyme dependent RNAi expression cassette, RIM3 RNAi hairpin structure insertion site, WPRE, bGHO (A) signal and AAV2ITR. Based on bioinformatics analysis, the invention discovers that the synapse transmission related protein Rab3-interacting molecule-3 (RIM 3 for short) which is highly expressed in the mouse pain model, so that a shRNA interference strategy is constructed, and the nociception of the pain model animal can be effectively relieved.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a RIM3-RNAi based on a Cre-lox recombination system and application thereof.
Background
Rna interference technology:
RNA interference (RNAi) refers to the phenomenon of highly conserved, highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. Since the expression of a specific gene in a cell or organism can be specifically reduced or shut down using an RNAi technology, the technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. A genetic mechanism for reverse regulation of gene expression has also been studied based on this phenomenon. In animals, RNAi can be achieved by U6-initiated expression of shRNA. Current U6 vectors can be expressed in animal cells by 2 means: transient expression and stable expression.
Cre-lox technique:
the Cre-lox technology is a site-specific recombination technology found in the 20 th century, 80 s from P1 phage. The technology can realize deletion, turnover, insertion, translocation and the like of the target fragment through the interaction of Cre recombinase and lox sites. The technology can act on DNA substrates with various structures, such as linear, annular and even supercoiled DNA, without any auxiliary factors, and is simple, quick and efficient to operate, so that the technology is widely applied to researches of gene knockout, insertion, turnover, translocation and the like of eukaryotes and prokaryotes. At present, the most popular field of application of the Cre-lox system is gene targeting, which has proven to be the most useful tool for genetic manipulation of mammalian cells and mice, and the combination of the system and gene targeting provides a means to achieve conditional gene knockout or activation.
3. Virus dependent gene recombination
The transgenic animal depends on gene recombination, which has the defects of long time consumption, high cost, low region or tissue specificity and the like. The Cre-lox system is used in a relatively flexible manner, for example by introducing the Cre or lox element by means of viruses, so that gene recombination is achieved in mice. By means of the cre-lox recombination system expressed by viruses, the labeling and manipulation of certain cells can be specific. When the structure and the function of a brain specific nerve loop are analyzed by the nerve tracing technology, the Cre recombinase system is combined with AAV serotypes (such as rAAV2/9, rAAV2/retro and rAAV 2/1) to achieve the aim of specifically researching the nerve loop marking and the function: the virus can ensure regional specific infection by local injection, and can realize stronger regional and cell specific gene recombination by adding a specific promoter for driving Cre genes. However, there is a need for the development of expression tools that involve a more optimal spatiotemporal specific regulation of important genes related to the function of a specific neural circuit with respect to how spatiotemporal specificity is disturbed.
Disclosure of Invention
The embodiment of the invention aims to provide RIM3-RNAi based on a Cre-lox recombination system and application thereof, and aims to solve the problems in the background technology.
The embodiment of the invention is realized in such a way that the RIM3-RNAi based on the Cre-lox recombination system comprises an RNAi expression vector of Cre enzyme-dependent rAAV expression, wherein the RNAi expression vector of Cre enzyme-dependent rAAV expression comprises the following components in sequence connected: AAV2ITR, promoter of excitatory neuronal expression CaMKII alpha, cre enzyme dependent RNAi expression cassette, RIM3 RNAi hairpin structure insertion site, WPRE, bGHO (A) signal and AAV2ITR.
According to a further technical scheme, the specific sequence of the RIM3 shRNA is as follows:
5’-GCCTGTGTGTGGATCTCAT-3’。
the specific sequence of the control group of the Scramble shRNA is as follows:
5’-GGTTTATATCGCGGTTATT -3’。
another object of the embodiment of the invention is the application of RIM3-RNAi based on Cre-lox recombination system, which is applied to the preparation of recombinant virus pAAV2-CaMKII alpha-DIO- (mCherry-bGH polyA-U6) -shRNA (RIM 3/Scramble) -WPRE-hGH polyA.
According to a further technical scheme, the recombinant virus pAAV2-CaMKII alpha-DIO- (mCherry-bGH polyA-U6) -shRNA (RIM 3/Scramble) -WPRE-hGH polyA is injected into the amygdala basalis of the mouse, helper virus AAV2/1-hSyn-Cre-EGFP is injected into the island leaves of the mouse, and after the virus is expressed for four weeks, the mechanical pain threshold of the sole of the mouse is observed after the total fibular nerve ligation model.
According to the RIM3-RNAi based on the Cre-lox recombination system and the application thereof provided by the embodiment of the invention, shRNA interference is carried out on specific brain Rab3-interacting molecule-3 (RIM 3 for short) molecules through the Cre-lox recombination system dependent on adeno-associated viruses, so that the RIM is applied to analgesic treatment. Based on bioinformatics analysis, the invention discovers that the high-expression synaptic transmission related protein RIM3 (which is a synaptic active protein and is positioned after synapses and participates in regulating exocytosis of presynaptic vesicles through acting on voltage-dependent calcium channels, and is expressed in a plurality of brain regions of a nervous system) in a mouse pain model, so that a shRNA interference strategy is constructed, nociception of pain model animals can be effectively relieved, and the invention provides a direction for targeted analgesic treatment.
Drawings
FIG. 1 is a graph showing RIM3-RNAi based on Cre-lox recombination system and the pain model mouse IC and BLA brain region RIM3 molecule expression control in application thereof according to the embodiment of the present invention.
FIG. 2 is a diagram of RIM3-RNAi based on Cre-lox recombination system and detection of RIM3 luciferase in application thereof according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of RIM3-RNAi based on Cre-lox recombination system and virus injection in application thereof according to an embodiment of the present invention.
FIG. 4 is a schematic diagram of a RIM3-RNAi based on a Cre-lox recombination system and a behavioral test result in application thereof according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
The RIM3-RNAi based on the Cre-lox recombination system provided by one embodiment of the invention comprises an RNAi expression vector of Cre enzyme-dependent rAAV expression, wherein the expression vector comprises the following components in sequence: AAV2ITR, promoter of excitatory neuronal expression CaMKII alpha, cre enzyme dependent RNAi expression cassette, RIM3 RNAi hairpin structure insertion site, WPRE, bGHO (A) signal and AAV2ITR.
The specific sequences of RIM3 shRNA used were: 5'-GCCTGTGTGTGGATCTCAT-3'; the specific sequence of the control group of the Scramble shRNA is as follows: 5'-GGTTTATATCGCGGTTATT-3'.
The specific steps of the mouse pain model experiment include:
step 1: a mouse fibular total nerve ligation (common peroneal nerve ligation, CPNL) model was established: after the mice are anesthetized by 2% isoflurane, the left leg is cut to expose the common fibular nerve, the common fibular nerve is partially sutured and disinfected after the aseptic operation line is ligated, and the animals are put back into a cage for feeding after being awake. The sham group only exposed the common fibular nerve, but did not ligate.
Step 2: mechanical pain detection: animals were preoperatively tested for mechanical pain threshold of the left foot using von-frey filaments (grams 0.008g,0.02g,0.04g,0.16g,0.4g,0.6g,1g,1.4g,2 g), recorded as day 0 data, and tested for mechanical pain threshold of the left foot at a fixed time daily beginning on day 1 after surgery, and statistically analyzed.
Step 3: western blot analysis: on day 7 post-operation animals were anesthetized to isolate brains, protein extraction was performed on the right IC and BLA brain regions, and semi-quantitative analysis was performed on protein expression such as RIM3 (results are shown in FIG. 1).
Step 4: virus construction and injection: construction of recombinant adeno-associated virus pAAV2-CaMKII alpha-DIO- (mCherry-bGH-polyA-U6) -shRNA (RIM 3/Scramble) -WPRE-hGH polyA and helper virus AAV2/1-hSyn-Cre-EGFP by the Wohangmi company were committed and the shRNA was verified in HEK293 cells as shown in FIG. 2. The virus was then injected on the right BLA (200 nl of virus titer>10 12 ) Helper virus was injected into the right IC of mice (200 nl in amount to ensure trans-synaptic, viral titre) with trans-synaptic AAV2/1-hSyn-Cre-EGFP>10 13 ) The injection method is shown in fig. 3.
Step 5: animal behavioural detection: after 4 weeks of virus expression, a fibular total nerve ligation model was established, the mechanical pain threshold of the left hind paw of the mice was detected, and statistical analysis was performed on the pain threshold of each group of animals, as shown in fig. 4: pain threshold of pain model animals injected with shRNA (RIM 3) is obviously increased, while pain threshold of pain model animals injected with shRNA (Scramble) is not obviously changed compared with that of pain model animals injected with no virus, RIM3 molecule expression on an IC-BLA nerve pathway is reduced through shRNA, and pain can be effectively relieved.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (2)
1. An application of Cre enzyme dependent rAAV expressed RNAi expression vector in preparing analgesic drugs, characterized in that Cre enzyme dependent rAAV expressed RNAi expression vector comprises the following components connected in sequence: AAV2ITR, promoter of excitatory neuronal expression CaMKII alpha, cre enzyme dependent RNAi expression cassette, rab3-interacting molecule-3 RNAi hairpin insertion site, WPRE, bGHO (A) signal and AAV2ITR.
2. The use of the Cre enzyme dependent rAAV expressed RNAi expression vector according to claim 1 in the preparation of analgesic drugs, wherein the specific sequence of Rab3-interacting molecule-3 shRNA used is:
5’-GCCTGTGTGTGGATCTCAT-3’。
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