中国神经再生研究(英文版) ›› 2023, Vol. 18 ›› Issue (8): 1827-1833.doi: 10.4103/1673-5374.358618

• 原著:神经损伤修复保护与再生 • 上一篇    下一篇

基于狂犬病病毒的高效逆行转导和逆行跨单突触示踪的工具病毒系统

  

  • 出版日期:2023-08-15 发布日期:2023-02-24
  • 基金资助:
    国家自然科学基金项目(32100899,31830035,31771156,21921004);国家科技创新2030项目(2021ZD0201003);广东省重点领域研发计划项目(2018B030331001);中国科学院战略优先研究计划项目(XDB32030200);深圳市生物医学病毒载体重点实验室项目(ZDSYS20200811142401005)

A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing

Kun-Zhang Lin1, 2, *, Lei Li2, Wen-Yu Ma2, 3, Xin Yang2, Zeng-Peng Han1, 2, 3, Neng-Song Luo1, 2, 4, Jie Wang2, 3, Fu-Qiang Xu1, 2, 3, 4, 5, 6, *   

  1. 1The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, Shenzhen, Guangdong Province, China; 2State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, Hubei Province, China; 3University of Chinese Academy of Sciences, Beijing, China; 4Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, Hubei Province, China; 5Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, Guangdong Province, China; 6Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.
  • Online:2023-08-15 Published:2023-02-24
  • Contact: Fu-Qiang Xu, PhD, fq.xu@siat.ac.cn; Kun-Zhang Lin, PhD, kz.lin@siat.ac.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, Nos. 32100899 (to KZL), 31830035 (to FQX), 31771156 (to FQX), 21921004 (to FQX); the National Science and Technology Innovation 2030, No. 2021ZD0201003 (to FQX); the Key-Area Research and Development Program of Guangdong Province, No. 2018B030331001 (to FQX); the Strategic Priority Research Program of the Chinese Academy of Sciences, No. XDB32030200 (to FQX); and the Shenzhen Key Laboratory of Viral Vectors for Biomedicine, No. ZDSYS20200811142401005 (to FQX).

摘要:

分析大脑神经网络的结构和功能是揭示大脑工作原理和脑部疾病发生机制的基础。重组狂犬病病毒载体可用于投射神经元的逆行标记以及细胞特异性跨单突触示踪,这使其成为标记突触输入的有力候选者。尽管已开发了多种减毒狂犬病病毒载体,但这些载体在功能网络研究中受到制备周期长和产量低的阻碍。为克服这些限制,此次实验开发了一种新型工具病毒生产系统,可快速拯救和制备高滴度CVS-N2c-ΔG病毒。且这种N2cG包被的CVS-N2c-ΔG能高效逆行访问rAAV9-Retro未及的投射神经元,效率是rAAV9-Retro的6倍。而密码子优化的嵌合型糖蛋白介导的CVS-N2c-ΔG病毒的跨单突触效率比密码子优化的嵌合型糖蛋白介导的SAD-B19-ΔG高2-3倍。CVS-N2c-ΔG可搭载功能探针用于神经活动监测,且可维持的时间窗口达3周,同时可表达足够的重组酶以进行有效的转基因重组。这种基于狂犬病病毒的高效逆行转导和逆行跨单突触示踪的工具病毒系统可作为研究神经回路的结构和功能的有力工具。

https://orcid.org/0000-0001-5091-6197 (Kun-Zhang Lin)

关键词: 神经回路, 狂犬病病毒, 投射神经元, 逆行转导, rAAV9-Retro, 跨单突触示踪, 突触输入, 转基因重组, 神经活动, 功能研究

Abstract: Analyzing the structure and function of the brain’s neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases. Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing, making these vectors powerful candidates for the dissection of synaptic inputs. Although several attenuated rabies viral vectors have been developed, their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors. To overcome these limitations, we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus. Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably: (1) N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro, and the efficiency was six times higher than that of rAAV9-Retro; (2) the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2–3 times higher than that of oG-mediated SAD-B19-ΔG; (3) CVS-N2c-ΔG could delivery modified genes for neural activity monitoring, and the time window during which this was maintained was 3 weeks; and (4) CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination. These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

Key words: functional studies, neural activity, neural circuits, projection neurons, rAAV9-Retro, rabies virus, recombination, retrograde labeling, synaptic inputs, trans-monosynaptic tracing