技术领域Technical field
本发明属于生物医学技术领域，尤其涉及一种基于Cre-lox重组系统的RIM3-RNAi及其应用。The invention belongs to the field of biomedical technology, and in particular relates to a RIM3-RNAi based on Cre-lox recombination system and its application.
背景技术Background technique
1.RNA干扰技术：1.RNA interference technology:
RNA干扰（RNA interference, RNAi）是指在进化过程中高度保守的、由双链RNA（double-stranded RNA，dsRNA）诱发的、同源mRNA高效特异性降解的现象。由于使用RNAi技术可以特异性降低或关闭特定基因在细胞或有机体中的表达，所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。人们也基于这一现象研究了一种反向调控基因表达的遗传学机制。在动物中，RNAi可以通过U6启动表达shRNA来实现。目前的U6载体可以通过2种方式表达在动物细胞内表达：瞬时表达和稳定表达。RNA interference (RNAi) refers to a phenomenon that is highly conserved during evolution, induced by double-stranded RNA (dsRNA), and causes efficient and specific degradation of homologous mRNA. Because RNAi technology can specifically reduce or turn off the expression of specific genes in cells or organisms, this technology has been widely used in the field of exploring gene functions and gene therapy for infectious diseases and malignant tumors. Based on this phenomenon, people have also studied a genetic mechanism that reversely regulates gene expression. In animals, RNAi can be achieved by U6-initiated expression of shRNA. The current U6 vector can be expressed in animal cells in two ways: transient expression and stable expression.
2.Cre-lox技术：2.Cre-lox technology:
Cre-lox技术是20世纪80年代从P1噬菌体中发现的一种位点特异性重组技术。该技术可通过Cre重组酶和lox位点的相互作用，实现目的片段的删除、翻转、插入和易位等。该技术不需要借助任何辅助因子，可作用于多种结构的DNA底物，如线形、环状甚至超螺旋DNA等，操作简单、快速、高效，因此被广泛应用于真核生物和原核生物的基因敲除、插入、翻转和易位等研究中。目前，Cre-lox系统应用最热门的领域是基因打靶，已被证明是哺乳动物细胞和小鼠遗传操作最有用的工具，该系统和基因打靶的结合提供了实现条件基因敲除或激活的手段。Cre-lox technology is a site-specific recombination technology discovered from P1 phage in the 1980s. This technology can achieve the deletion, flipping, insertion and translocation of the target fragment through the interaction between Cre recombinase and lox site. This technology does not require any auxiliary factors and can act on DNA substrates of various structures, such as linear, circular and even superhelical DNA. It is simple, fast and efficient to operate, so it is widely used in eukaryotes and prokaryotes. Research on gene knockout, insertion, flipping and translocation. Currently, 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. The combination of the system and gene targeting provides a means to achieve conditional gene knockout or activation. .
3.病毒依赖的基因重组3. Virus-dependent genetic recombination
由于转基因动物依赖的基因重组存在耗时长、成本高、区域或者组织特异性不高等不足。采用比较灵活的方式使用Cre-lox系统，例如通过病毒引入Cre或lox元件，从而在小鼠中实现基因重组。借助病毒表达的cre-lox重组系统，可以特异性对某些细胞的标记和操控。当前运用神经示踪技术对大脑特定神经环路的结构和功能进行解析时，运用 Cre 重组酶系统和 AAV 血清型（比如 rAAV2/9、rAAV2/retro、rAAV2/1）结合，达到特异性对神经环路标记和功能研究的目的：病毒可以通过局部注射的方式保证区域特异性感染，再加上驱动 Cre 基因的特异性启动子，能够实现更强的区域和细胞特异性的基因重组。但是对于如何时空特异性地干扰特定的神经环路的功能，需要涉及一种更优的时空特异调控相关重要基因的表达工具的开发。The genetic recombination that transgenic animals rely on has shortcomings such as long time consumption, high cost, and low regional or tissue specificity. The Cre-lox system is used in a more flexible way, such as introducing Cre or lox elements through viruses to achieve genetic recombination in mice. With the help of the virally expressed cre-lox recombination system, certain cells can be specifically marked and manipulated. When neurotracing technology is currently used to analyze the structure and function of specific neural circuits in the brain, the Cre recombinase system is used to combine with AAV serotypes (such as rAAV2/9, rAAV2/retro, rAAV2/1) to achieve specificity on neural circuits. The purpose of circuit labeling and functional studies: The virus can ensure region-specific infection through local injection, and coupled with the specific promoter driving the Cre gene, stronger region- and cell-specific gene recombination can be achieved. However, how to specifically interfere with the function of specific neural circuits in a spatiotemporal manner requires the development of a better expression tool for spatiotemporally specific regulation of relevant important genes.
发明内容Contents of the invention
本发明实施例的目的在于提供一种基于Cre-lox重组系统的RIM3-RNAi及其应用，旨在解决背景技术中提出的问题。The purpose of the embodiments of the present invention is to provide a RIM3-RNAi based on the Cre-lox recombination system and its application, aiming to solve the problems raised in the background technology.
本发明实施例是这样实现的，一种基于Cre-lox重组系统的RIM3-RNAi，包括一种Cre酶依赖的rAAV表达的RNAi表达载体，所述的Cre酶依赖的rAAV表达的RNAi表达载体包括顺次连接的：AAV2ITR，兴奋性神经元表达的启动子CaMKIIα，Cre酶依赖的RNAi表达盒，RIM3RNAi发卡结构插入位点，WPRE， bGHpoly（A）signal以及AAV2ITR。The embodiment of the present invention is implemented as follows. A RIM3-RNAi based on the Cre-lox recombination system includes an RNAi expression vector for Cre enzyme-dependent rAAV expression. The RNAi expression vector for Cre enzyme-dependent rAAV expression includes Sequentially connected: AAV2ITR, excitatory neuron expression promoter CaMKIIα, Cre enzyme-dependent RNAi expression cassette, RIM3RNAi hairpin structure insertion site, WPRE, bGHpoly(A) signal and AAV2ITR.
进一步的技术方案，所使用的RIM3 shRNA 的具体序列为：For further technical solutions, the specific sequence of RIM3 shRNA used is:
5’-GCCTGTGTGTGGATCTCAT-3’。5’-GCCTGTGTGTGGATCTCAT-3’.
对照组Scramble shRNA的具体序列为：The specific sequence of Scramble shRNA in the control group is:
5’-GGTTTATATCGCGGTTATT -3’。5’-GGTTTATATCGCGGTTATT-3’.
本发明实施例的另一目的在于，一种基于Cre-lox重组系统的RIM3-RNAi的应用，将所述的RIM3-RNAi应用于重组病毒pAAV2-CaMKIIα-DIO-(mCherry-bGH polyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA的制备。Another object of the embodiment of the present invention is to provide an application of RIM3-RNAi based on the Cre-lox recombination system, and apply the RIM3-RNAi to the recombinant virus pAAV2-CaMKIIα-DIO-(mCherry-bGH polyA-U6) -Preparation of shRNA(RIM3/Scramble)-WPRE-hGH polyA.
进一步的技术方案，将所述的重组病毒pAAV2-CaMKIIα-DIO-(mCherry-bGHpolyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA 注射于小鼠基底外侧杏仁核，并将辅助病毒AAV2/1-hSyn-Cre-EGFP 注射到小鼠岛叶，待病毒表达四周后，观察到腓总神经结扎模型小鼠后足底机械痛阈值。A further technical solution is to inject the recombinant virus pAAV2-CaMKIIα-DIO-(mCherry-bGHpolyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA into the basolateral amygdala of mice, and add the helper virus AAV2 /1-hSyn-Cre-EGFP was injected into the insula of mice, and four weeks after virus expression, the mechanical pain threshold of the plantar foot of common peroneal nerve ligation model mice was observed.
本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用，通过腺相关病毒依赖的Cre-lox重组系统，对特定脑区Rab3-interacting molecule-3 (简称RIM3)分子进行shRNA干扰，从而应用于镇痛治疗。本发明在生物信息学分析的基础上，发现小鼠痛模型中高表达的突触传递相关蛋白RIM3（是一种突触活性蛋白，位于在突触后，通过作用于电压依赖性的钙通道，参与调节突触前囊泡的胞吐作用，在神经系统的多个脑区都有表达），因此构建了shRNA干扰策略，可有效缓解痛模型动物的伤害性感受，本发明将为靶向镇痛治疗提供方向。The embodiments of the present invention provide a kind of RIM3-RNAi based on the Cre-lox recombination system and its application. Through the adeno-associated virus-dependent Cre-lox recombination system, Rab3-interacting molecule-3 (RIM3) molecules in specific brain areas are carried out. shRNA interference for analgesic treatment. On the basis of bioinformatics analysis, the present invention found that the synaptic transmission-related protein RIM3 (which is a synaptic active protein located at the postsynapsis and acts on voltage-dependent calcium channels) was highly expressed in the mouse pain model. Involved in regulating the exocytosis of presynaptic vesicles and expressed in multiple brain areas of the nervous system), an shRNA interference strategy has been constructed that can effectively alleviate the nociception of pain model animals. The present invention will provide targeted analgesia. Pain treatment provides direction.
附图说明Description of the drawings
图1为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的痛模型小鼠IC和BLA脑区RIM3分子表达对照图。Figure 1 is a comparison chart of RIM3 molecule expression in the IC and BLA brain regions of pain model mice using RIM3-RNAi based on the Cre-lox recombinant system and its application according to the embodiment of the present invention.
图2为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的RIM3荧光素酶检测图。Figure 2 is a RIM3-RNAi based on the Cre-lox recombination system and a RIM3 luciferase detection chart in its application provided by the embodiment of the present invention.
图3为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的病毒注射示意图。Figure 3 is a schematic diagram of RIM3-RNAi based on the Cre-lox recombination system and its application of virus injection provided by an embodiment of the present invention.
图4为本发明实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi及其应用中的行为学检测结果示意图。Figure 4 is a schematic diagram of the behavioral detection results of RIM3-RNAi based on the Cre-lox recombination system and its application provided by the embodiment of the present invention.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.
以下结合具体实施例对本发明的具体实现进行详细描述。The specific implementation of the present invention will be described in detail below with reference to specific embodiments.
本发明一个实施例提供的一种基于Cre-lox重组系统的RIM3-RNAi，包括一种Cre酶依赖的rAAV表达的RNAi表达载体，该表达载体包含顺次连接的：AAV2ITR，兴奋性神经元表达的启动子CaMKIIα，Cre酶依赖的RNAi表达盒，RIM3 RNAi发卡结构插入位点，WPRE，bGHpoly（A）signal以及AAV2ITR。One embodiment of the present invention provides a RIM3-RNAi based on the Cre-lox recombination system, including an RNAi expression vector for Cre enzyme-dependent rAAV expression. The expression vector includes sequentially connected: AAV2ITR, excitatory neuron expression The promoter of CaMKIIα, Cre enzyme-dependent RNAi expression cassette, RIM3 RNAi hairpin insertion site, WPRE, bGHpoly(A) signal, and AAV2ITR.
所使用的RIM3 shRNA 的具体序列为：5’-GCCTGTGTGTGGATCTCAT-3’；对照组Scramble shRNA的具体序列为：5’-GGTTTATATCGCGGTTATT -3’。The specific sequence of the RIM3 shRNA used is: 5’-GCCTGTGTGTGGATCTCAT-3’; the specific sequence of the control Scramble shRNA is: 5’-GGTTTATATCGCGGTTATT-3’.
小鼠痛觉模型实验的具体步骤包括：The specific steps of the mouse pain model experiment include:
步骤1：建立小鼠腓总神经结扎（common peroneal nerve ligation，CPNL）模型：小鼠通过2%异氟醚麻醉后，在左腿做切口暴露腓总神经，无菌手术线结扎腓总神经后局部缝合消毒，待动物清醒后放回笼中饲养。假手术组仅暴露腓总神经，但不进行结扎。Step 1: Establish a common peroneal nerve ligation (CPNL) model in mice: After the mice are anesthetized with 2% isoflurane, an incision is made in the left leg to expose the common peroneal nerve, and the common peroneal nerve is ligated with sterile surgical thread. The local area was sutured and disinfected, and the animals were returned to the cage for rearing after they woke up. In the sham operation group, only the common peroneal nerve was exposed without ligation.
步骤2：机械痛检测：动物手术前使用von-frey丝（克数为0.008g，0.02g，0.04g，0.16g，0.4g，0.6g，1g，1.4g，2g）检测左足机械痛阈值，记为第0天数据，手术后从第1天开始，每天固定时间检测左足机械痛阈值，并进行统计学分析。Step 2: Mechanical pain detection: Use von Frey wire (grams are 0.008g, 0.02g, 0.04g, 0.16g, 0.4g, 0.6g, 1g, 1.4g, 2g) to detect the mechanical pain threshold of the left foot before animal surgery. The data was recorded as the 0th day. Starting from the 1st day after the operation, the mechanical pain threshold of the left foot was measured at a fixed time every day, and statistical analysis was performed.
步骤3：蛋白印迹分析：动物术后第7天，麻醉分离大脑，对右侧IC和BLA脑区进行取材提蛋白，对RIM3等蛋白表达进行半定量分析（结果如图1所示）。Step 3: Western blot analysis: On the 7th day after the operation, the animals were anesthetized and the brains were isolated. The right IC and BLA brain areas were sampled to extract proteins, and the expression of RIM3 and other proteins was semi-quantitatively analyzed (the results are shown in Figure 1).
步骤4：病毒构建与注射：委托武汉枢密公司构建重组腺相关病毒pAAV2-CaMKIIα-DIO-(mCherry-bGH-polyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA 和辅助病毒AAV2/1-hSyn-Cre-EGFP，在HEK293细胞对shRNA进行验证，结果如图2所示。随后将该病毒注射在小鼠右侧BLA (注射量为200nl，病毒滴度>1012)，将辅助病毒跨突触AAV2/1-hSyn-Cre-EGFP注射在小鼠右侧IC(注射量为200nl，为了确保跨突触，病毒滴度>1013)，注射方法如图3所示。Step 4: Virus construction and injection: Wuhan Privy Company was entrusted to construct the recombinant adeno-associated virus pAAV2-CaMKIIα-DIO-(mCherry-bGH-polyA-U6)-shRNA(RIM3/Scramble)-WPRE-hGH polyA and the helper virus AAV2/1 -hSyn-Cre-EGFP, shRNA was verified in HEK293 cells, and the results are shown in Figure 2. The virus was then injected into the right BLA of the mouse (injection volume: 200nl, virus titer >1012 ), and the helper virus trans-synaptic AAV2/1-hSyn-Cre-EGFP was injected into the right IC of the mouse (injection volume: For 200 nl, to ensure trans-synaptic viral titer >1013 ), the injection method is shown in Figure 3.
步骤5：动物行为学检测：病毒表达4周后，建立腓总神经结扎模型，检测小鼠左后足机械痛阈值，对各组动物痛阈进行统计学分析，如图4所示：注射shRNA（RIM3）的痛模型动物痛阈明显上升，而注射shRNA（Scramble）的痛模型动物痛阈相比于无病毒注射组没有明显改变，通过shRNA降低IC-BLA神经通路上的RIM3分子表达，可以有效镇痛。Step 5: Animal behavior test: 4 weeks after virus expression, establish a common peroneal nerve ligation model, detect the mechanical pain threshold of the left hind foot of mice, and perform statistical analysis on the pain threshold of animals in each group, as shown in Figure 4: Injection of shRNA (RIM3) pain model animals' pain threshold increased significantly, while the pain threshold of pain model animals injected with shRNA (Scramble) did not change significantly compared with the virus-free injection group. By reducing the expression of RIM3 molecules on the IC-BLA neural pathway through shRNA, it can Effective analgesia.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919676A (en)* | 1993-06-24 | 1999-07-06 | Advec, Inc. | Adenoviral vector system comprising Cre-loxP recombination |
| CN101460634A (en)* | 2006-04-13 | 2009-06-17 | 康乃尔研究基金会有限公司 | Methods and compositions for targeting C-REL |
| CN104093830A (en)* | 2011-04-15 | 2014-10-08 | 吉恩勒克斯公司 | Cloned strains of attenuated vaccinia virus and methods of use thereof |
| CN104736166A (en)* | 2012-05-30 | 2015-06-24 | 哈佛大学校长及研究员协会 | Engineered Botulinum Neurotoxin |
| CN107630009A (en)* | 2016-07-19 | 2018-01-26 | 中国科学院武汉物理与数学研究所 | It is a kind of to be attenuated blast, replicate controllable HSV recombinant viruses and preparation method and application |
| CN109476713A (en)* | 2016-06-08 | 2019-03-15 | 儿童医学中心公司 | Engineered botulinum neurotoxin |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2560897A (en)* | 2017-03-23 | 2018-10-03 | Lotvall Jan | Tissue-derived extracellular vesicles and their use as diagnostics |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919676A (en)* | 1993-06-24 | 1999-07-06 | Advec, Inc. | Adenoviral vector system comprising Cre-loxP recombination |
| CN101460634A (en)* | 2006-04-13 | 2009-06-17 | 康乃尔研究基金会有限公司 | Methods and compositions for targeting C-REL |
| CN104093830A (en)* | 2011-04-15 | 2014-10-08 | 吉恩勒克斯公司 | Cloned strains of attenuated vaccinia virus and methods of use thereof |
| CN104736166A (en)* | 2012-05-30 | 2015-06-24 | 哈佛大学校长及研究员协会 | Engineered Botulinum Neurotoxin |
| CN109476713A (en)* | 2016-06-08 | 2019-03-15 | 儿童医学中心公司 | Engineered botulinum neurotoxin |
| CN107630009A (en)* | 2016-07-19 | 2018-01-26 | 中国科学院武汉物理与数学研究所 | It is a kind of to be attenuated blast, replicate controllable HSV recombinant viruses and preparation method and application |
| Title |
|---|
| Glutamatergic synapses from the insular cortex to the basolateral amygdala encode observational pain;Ming-Ming Zhang et al;《Neuron》;第第110卷卷;第1-16页* |
| Publication number | Publication date |
|---|---|
| CN114410683A (en) | 2022-04-29 |
| Publication | Publication Date | Title |
|---|---|---|
| El-Shamayleh et al. | Strategies for targeting primate neural circuits with viral vectors | |
| Ravindra Kumar et al. | Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types | |
| Griffin et al. | Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction in the rat spinal cord following injury | |
| Mathiesen et al. | CNS transduction benefits of AAV-PHP. eB over AAV9 are dependent on administration route and mouse strain | |
| Duan et al. | The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model | |
| Chen et al. | Reactivation of dormant relay pathways in injured spinal cord by KCC2 manipulations | |
| JP7094236B2 (en) | Compositions, methods and uses for mutant AAV and gene transfer into cells, organs and tissues | |
| Rincon et al. | Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP. B vector | |
| Masamizu et al. | Local and retrograde gene transfer into primate neuronal pathways via adeno-associated virus serotype 8 and 9 | |
| Yu et al. | Intraganglionic AAV6 results in efficient and long-term gene transfer to peripheral sensory nervous system in adult rats | |
| Xu et al. | In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery | |
| Qiu et al. | Lighting up neural circuits by viral tracing | |
| Klaw et al. | Intraspinal AAV injections immediately rostral to a thoracic spinal cord injury site efficiently transduces neurons in spinal cord and brain | |
| CN111150854A (en) | A way to improve social disorder in autism | |
| Keswani et al. | Pseudotyped adeno‐associated viral vector tropism and transduction efficiencies in murine wound healing | |
| Baatartsogt et al. | Successful liver transduction by re‐administration of different adeno‐associated virus vector serotypes in mice | |
| An et al. | In vivo base editing extends lifespan of a humanized mouse model of prion disease | |
| CN114410683B (en) | A kind of RIM3-RNAi based on Cre-lox recombination system and its application | |
| Boehm et al. | Translational PET applications for brain circuit mapping with transgenic neuromodulation tools | |
| Inquimbert et al. | Stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord | |
| Pollack | A powerful new way to edit DNA | |
| Serrano et al. | Simple and highly specific targeting of resident microglia with adeno-associated virus | |
| CN114369623B (en) | A kind of Synaptotagmin2-RNAi based on Cre-lox recombination system and its application | |
| Grinevich et al. | Somatic transgenesis (viral vectors) | |
| Shi et al. | Research techniques made simple: delivery of the CRISPR/Cas9 components into epidermal cells |
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