显微注射病毒至小鼠脊髓背角的腰膨大部

Zhi-Jun Zhang; Peng-Bo Jing; Yong-Jing Gao

doi:10.21769/BioProtoc.2020

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Peer-reviewed

Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice

显微注射病毒至小鼠脊髓背角的腰膨大部

ZZ Zhi-Jun Zhang ^*

PJ Peng-Bo Jing ^*

YG Yong-Jing Gao email

(*contributed equally to this work) 发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2020 浏览次数: 15268

评审: Jia LiZhen ShiAnonymous reviewer(s)

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Cover of The Journal of Clinical Investigation, featuring study using the protocol.

Feb 2016

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Abstract

In order to explore the role of a specific gene/protein in the specific segment of the spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, lentivirus containing shRNA for CXCL13 were injected into the dorsal horn of the spinal lumbar enlargement segment (Jiang et al., 2016). This technique allows the study of the role of CXCL13 in the ipsilateral dorsal horn in neuropathic pain without affecting DRG or contralateral dorsal horn.

Background

The dorsal horn of spinal cord is a well-organized and stratified neuronal complex, which transmits the sensory information from the body surface and deep tissues to the brain. The activity of neurons in the spinal dorsal horn can be regulated by primary afferents, descending fibers, as well as spinal glial cells such as astrocytes and microglia. Understanding how the specific protein and gene in spinal neurons or glial cells contribute to the mechanism of physiological and pathological pain is of increasing interest. In order to explore the role of a specific gene/protein in the spinal cord, the technique of intrathecal injection is commonly used to deliver agonist, antagonist, siRNA, or miRNA into the subarachnoid space. The knockout and conditional deletion of a specific gene is another exhaustive and expensive approach. However, the limitation of these methods is lack of spinal segment- and cell type-specific deletion or overexpression. This protocol shows that intraspinal injection can deliver specific viral vectors into specific segmental spinal dorsal horn to investigate the function of a gene in a region- and cell type-specific manner.

Materials and Reagents

0.2 ml sterile centrifuge tubes (Corning, Axygen^®, catalog number: PCR-02-C )
10 μl sterile tips (Corning, Axygen^®, catalog number: T-300L )
Glass capillary (World Precision Instruments, catalog number: TW150F-4 )
10 μl microsyringe (Shanghai Gaoge Industrial and Trading)
Insulin syringe, 30 G (BD, catalog number: 1312073 )
4-0 silk (Ethicon)
Aseptic gauze
Cotton swabs
Hot glue (Shanghai Santo, catalog number: 1625 )
ICR mice, male, 8 weeks old
LV-CXCL13 shRNA
Note: The recombinant lentivirus containing CXCL13 shRNA was packaged using pGCSIL-GFP vector by Shanghai GeneChem Lentiviral vector. (CXCL13, GenBank Accession number: NM_018866).
Liquid paraffin (EMD Millipore, catalog number: 107162 )
Enhanced infection solution (Shanghai GeneChem, catalog number: REVG0002 )
Note: This is a gift product with lentivirus. The website does not show it as a product that can be purchased
Pentobarbital sodium (Sigma-Aldrich, catalog number: P3761 )
Ethanol, pure (Sigma-Aldrich, catalog number: E7023 )
1% povidone iodine (Shanghai Bangshili disinfectant)
Phosphate buffered saline (1x PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
4% paraformaldehyde
Picric acid

Equipment

Model P-97 flaming micropipette puller (Sutter instrument, model: P-97 )
Microscissors (Fine Science Tools, catalog number: 91500-09 )
Microforceps (Fine Science Tools, catalog number: 91150-20 )
Surgery microscope (RWD Life Science, catalog number: 77019 )
Heating pad (Yiwu pet, Small size)
Biological safety cabinet (Thermo Fisher Scientific, mode: 1388 )
Pet trimmer (Wahl Home, catalog number: 58563 )
Desktop digital stereotaxic instruments (RWD Life Science, catalog number: 68025 )
Spinal clamp apparatus (RWD Life Science, catalog number: 68091 )
Hot glue gun (Shanghai Santo, catalog number: 1628 )
A set of surgery instruments for mouse (RWD Life Science, catalog number: SP0010-B )
Skull drill (RWD Life Science, catalog number: 78001 )
Legato syringe pumps (KD Scientific, catalog number: 788130 )

Procedure

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如何引用

Zhang, Z., Jing, P. and Gao, Y. (2016). Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice. Bio-protocol 6(22): e2020. DOI: 10.21769/BioProtoc.2020.