Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Summary

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Abstract

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2^fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

Introduction

Modern studies of the structure and function of the brain usually require genetic manipulation of specific genes in neuronal cells. To probe the functions of different genes, transgenic mice carrying mutant alleles, including knockout and knock-in alleles have been routinely generated. Stereotaxic brain surgery for adult rodents is a standard method to locally deliver drugs, viruses, tracers and other reagents to specific regions of rodent brains^1,2. Applying the stereotaxic brain surgery to transgenic mice permits one to genetically manipulate the gene function and neuronal activity in specific neuronal popul....

Protocol

The animal protocols described here have been approved by the Animal Care and Use Committees of National Yang-Ming University.

1. Preparation of The Holder for Neonatal Pups in The Stereotaxic Apparatus

1. Make the head tray: cut the bottom of a 1.5 mL centrifuge tube (15 mm long) into the shape that fits the head of neonatal pups by removing 1/5 of the wall of the tube.
2. Take a pipette tip box with the right size that fits with the pedestal of stereotaxic apparatus, and remove the top cover. Affix the head tray prepared in step 1.1 and a tissue embedding cassette onto the base of tip tray with hot-melting adhesive. The he....

Results

For the first set of experiment, we microinjected 200 nL of AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 viruses (AAV-eGFP-Cre, 1/10 dilution in Dulbecco's phosphate buffered saline) that express the Cre DNA recombinase fused with GFP into P2 striatum of Ai14 mice. The Ai14 mice express tdTomato reporter gene upon Cre-mediated deletion of loxP-flanked (floxed) STOP cassette (Figure 2F). The brains were harvested at P14 for immunostaining of GFP and tdTomato. Many AAV.......

Discussion

In the present study, we demonstrate a simple and reliable stereotaxic method for injecting AAV viruses into the striatum of neonatal mouse brains. We microinjected AAV-eGFP-Cre viruses into the striatum of Ai14 reporter mice at P2 and then analyzed the reporter gene expression at P14. We found AAV transduced GFP-positive cells throughout the striatum at rostrocaudal levels. Moreover, nearly all GFP-positive cells co-expressed the tdTomato reporter gene in striatal cells, suggesting a successful Cre-loxP DNA recombinatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30G PrecisionGlide Needle	Becton Dickinson	REF 305106
Chloroform	JT Baker	9180-03
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	0.1%
Standard Stereotaxic Instruments	RWD Life Science	68037	Without using 68030 Mouse/Neonatal Rat Adaptor
Anti-FOXP2 antibody	Abcam	ab16046	Rabbit polyclonal to FOXP2, 1:4,000
Anti-RFP antibody	Abcam	ab65856	Mouse monoclonal to RFP, 1:1,000
BX63 microscope	Olympus	BX63
LSM 880 confocal microscope	Zeiss	LSM 880
Goat anti-rabbit conjugated Alexa fluor594	Jackson lmmunoReserch Laboratories Inc.	111-585-003
AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40	Penn Vector Core	AV-9-PV1848	Lot # CS0987, 5.506x1013 (GC/mL)
AAV9.chicken actin-eGFP	AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan	N/A	1x1014 (GC/ml)
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	The Jackson Labtorary	007914	Ai14
B6(Cg)-Foxp2tm1.1Sfis/CfreJ	The Jackson Labtorary	026259	Foxp2fl/fl
Dulbecco’s phosphate buffered saline	Corning cellgro	21-030-CVR