Stereotactic Microinjection of a Viral Vector for Gene Manipulation in a Mouse Pup Striatum

Abstract

Source: Chen, S., et al., Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains. J. Vis. Exp. (2018)

This video demonstrates the stereotaxic injection of a viral vector encoding Cre recombinase and green fluorescence protein or GFP into the striatum of a genetically modified mouse pup to achieve gene manipulation by excising a loxP-flanked STOP cassette, activating red fluorescence protein or RFP as a reporter.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Microinjection

1. Prepare sterile surgery by wiping the stereotaxic instrument thoroughly with 70% ethanol. Immerse the surgical instrument in 70% ethanol to sterilize it.
2. Scrub the pup's head with 70% ethanol. Locate the landmark lambda on the skull and mark the lambda with a marker pen. Aim the needle tip at the lambda, and set the anterior-posterior (AP) and medial-lateral (ML) coordinates as zero.
3. Move the injection arm to the target site according to the X and Y coordinates of the target site. For the striatum of postnatal day (P) 2 pups, the coordinates are: AP, +2.4 mm anterior to the lambda; ML, ±1.0 mm lateral from the midline; dorsal-ventral (DV), −1.7 mm from the skull. Mark the position of the fast green dye in the PE10 tube with a pen.
4. Bring down the 30G injection needle slowly to penetrate through the skin and skull, and then bring up the needle tip until it stops at the surface of the skull. Set the DV coordinate to zero.
5. Slowly bring down the 30G injection needle until it reaches the DV coordinate of the target site. Wait for 1 min to allow the parenchyma to resume its normal shape. Then, run the microinjection program (100 nL/min).
6. Make sure that the mark of fast green dye is moving in the PE tube to ensure the virus liquid is injected into the brain.
7. Wait for 1 min after the termination of the microinjection, and then slowly and progressively bring up the needle to 1/2 the height of the DV depth within 30 s. After 30 s, slowly withdraw the needle from the pup's head.

2. Post-surgical Recovery of The Pup

1. Warm up the pup for 20 min in a 33 °C incubator. Check the recovery of the pup from hypothermia anesthesia every 5 min until the pup has regained sufficient consciousness to maintain sternal recumbency.
2. Return the pup to the dam after the pup is fully recovered.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30G PrecisionGlide Needle	Becton Dickinson	REF 305106
Hamilton MICROLITER Syringe	Hamilton	80300	30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor
Polyethylene tubing PE20	Becton Dickinson	427406
Polyethylene tubing PE10	Becton Dickinson	427401
Micro Flow Rate Syringe Pump	Longer Precision Pump Co.	TJ-2A (Controller) and L0107-2A (Drive Unit)
25G syringe	Becton Dickinson	REF 302105
Fast green	Sigma-Aldrich	F-7252	0.10%
Standard Stereotaxic Instruments	RWD Life Science	68037	Without using 68030 Mouse/Neonatal Rat Adaptor
LSM 880 confocal microscope	Zeiss	LSM 880
AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40	Penn Vector Core	AV-9-PV1848	Lot # CS0987, 5.506x1013 (GC/mL)
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	The Jackson Labtorary	7914	Ai14
B6(Cg)-Foxp2tm1.1Sfis/CfreJ	The Jackson Labtorary	26259	Foxp2fl/fl