CN114369623B - Syntagmin 2-RNAi based on Cre-lox recombination system and application thereof - Google Patents
Syntagmin 2-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 Syntagmin 2-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, syt2 RNAi hairpin insertion site, WPRE, bGHO (A) signal and AAV2ITR. Based on bioinformatics analysis, the invention discovers that the synapse transmission related protein Syntagmin 2 (Syt 2 for short) is highly expressed in the mouse pain model, so that a shRNA interference strategy is constructed, nociception of the pain model animal can be effectively relieved, and the invention provides a direction for targeted analgesic treatment.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a Syntagmin 2-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 during evolution, induced by double-stranded RNA (dsRNA), and highly efficient and specific degradation of homologous mRNA. 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. Stable expression is achieved mainly by insertion into the genome.
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 Cre or loxP elements via viruses, so that gene recombination is achieved. 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 a Syntagmin 2-RNAi based on a Cre-lox recombination system and application thereof, and aims to solve the problems set forth in the background technology.
The embodiment of the invention is realized in such a way that the Syntagmin 2-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, syt2 RNAi hairpin insertion site, WPRE, bGHO (A) signal and AAV2ITR.
According to a further technical scheme, the specific sequence of the Syt2 shRNA is as follows:
5’-CCCTTTGACCCTCAGTGAT-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 Sydaptatami 2-RNAi based on the Cre-lox recombination system, wherein the Sydaptatami 2-RNAi is applied to the preparation of recombinant virus pAAV2-CaMKII alpha-DIO- (mCherry-bGH polyA-U6) -shRNA (Syt 2/Scramble) -WPRE-hGH polyA.
According to a further technical scheme, the recombinant virus pAAV2-CaMKII alpha-DIO- (mCherry-bGH polyA-U6) -shRNA (Syt 2/Scramble) -WPRE-hGH polyA is injected into island leaves of a mouse, helper virus AAV2/R-hSyn-cre-WPRE-hGH polyA is injected into amygdala basalis of the outer side of the mouse, and after the virus is expressed for four weeks, the rear plantar mechanical pain threshold of a mouse of a fibular total nerve ligation model is observed.
The Syntagmin 2-RNAi based on the Cre-lox recombination system and the application thereof provided by the embodiment of the invention are used for carrying out shRNA interference on specific brain region Syntagmin 2 molecules through the Cre-lox recombination system dependent on adeno-associated viruses, so as to be applied to analgesic treatment. Based on bioinformatics analysis, the invention discovers that a synapse transmission related protein Syntagmin 2 (Syt 2 for short) which is highly expressed in a mouse pain model is calcium binding protein which is helpful for calcium ions to trigger the release of a rapid neurotransmitter of central and neuromuscular synapses and is expressed in a plurality of brain regions of a nervous system, 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 shows synapttagmin 2-RNAi based on Cre-lox recombination system and pain model mouse IC and BLA brain region Syt2 molecule expression in application thereof according to the embodiment of the invention.
FIG. 2 is a diagram showing a Syt2 luciferase assay of Syaptotagmin 2-RNAi based on a Cre-lox recombination system and application thereof according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of a Syntagmin 2-RNAi based on a Cre-lox recombination system and virus injection in application thereof according to an embodiment of the present invention.
FIG. 4 shows a synapttagmin 2-RNAi based on a Cre-lox recombination system and a behavioral detection 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 invention provides a Syntagmin 2-RNAi based on a Cre-lox recombination system, which 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, syt2 RNAi hairpin insertion site, WPRE, bGHO (A) signal and AAV2ITR.
The specific sequences of the Syt2 shRNA used are: 5'-CCCTTTGACCCTCAGTGAT-3', specific sequences of control Scramble shRNA are: 5'-GGTTTATATCGCGGTTATT-3'.
The specific steps of the mouse pain model experiment include:
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.
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 and 2 g), recorded as day 0 data, tested for mechanical pain threshold of the left foot at a fixed time daily beginning on day 1 after surgery, and statistically analyzed.
3. Western blot analysis: on day 7 post-operation animals, brains were anesthetized and isolated, protein extraction was performed on the right IC and BLA brain regions, and semi-quantitative analysis was performed on protein expression of Syt2 and the like (results are shown in fig. 1).
4. Virus construction and injection: the recombinant adeno-associated virus pAAV2-CaMKII alpha-DIO- (mCherry-bGH-polyA-U6) -shRNA (Syt 2/Scramble) -WPRE-hGH polyA and helper virus were constructed by the trusted Whan Pivot company, and the shRNA was verified in HEK293 cells, and the results are shown in FIG. 2. The virus was then injected into the right IC (200 nl of virus titer>10 12 ) Helper virus AAV2/R-hSyn-cre-WPRE-hGH polyA was injected at the right BLA (200 nl injection, viral titer>10 12 ) The injection method is shown in fig. 3.
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: the pain threshold of the pain model animal injected with shRNA (Syt 2) is obviously increased, while the pain threshold of the pain model animal injected with shRNA (Scramble) is not obviously changed compared with that of a virus-free injection group, and the pain can be effectively relieved by reducing the expression of Syt2 molecules on an IC-BLA nerve pathway through shRNA.
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, syntagmin 2 RNAi hairpin structure 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 the used synatagmin 2 shRNA is:
5’-CCCTTTGACCCTCAGTGAT-3’。
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| GB2560897A (en) * | 2017-03-23 | 2018-10-03 | Lotvall Jan | Tissue-derived extracellular vesicles and their use as diagnostics |
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