A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

Summary

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

Abstract

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

Introduction

Genome-wide association studies (GWAS) have driven the discovery of novel candidate genes that are associated with brain pathologies^1,2,3,4. These studies have been particularly beneficial in understanding devastating neuropsychiatric disorders such as schizophrenia (SCZ), where the investigation of novel genes has served as a launching point for new lines of research and therapeutic intervention^5,6. Genes harboring risk for SCZ show biased expression in the prefrontal cortex (PFC) d....

Protocol

All experimental protocols were conducted according to the National Institutes of Health (NIH) guidelines for animal research and were approved by the Boston University Institutional Animal Care and Use Committee (IACUC).

1. DNA solution preparation

1. Purchase a commercial plasmid or subclone a gene of interest into plasmid with desired promoter. Here, a plasmid containing EGFP under the CAG promoter (pCAG-EGFP) was used.
   NOTE: Determine the desired promoter based off the leve.......

Discussion

Herein, a pipeline is described that combines the manipulation of novel genes of interest in large populations of frontal cortical neurons with behavioral assays in mice. Moreover, this pipeline allows for the longitudinal study of behavior in the same mice both during early postnatal development and in adulthood. This technique bypasses the need to rely on genetic animal models that can be costly in terms of time and expenses. The strength of this protocol is that it can be used to study neurodevelopmental and neuropsyc.......

Materials

Name	Company	Catalog Number	Comments
13mm Silk Black Braided Suture	Havel's	SB77D	Suture skin
Adson Forceps	F.S.T.	11006-12	IUE
C270 Webcam	Logitech	N/A	Record behavior
Electroporator	Custom-built	N/A	See Figure 1 and 2 and Bullmann et al, 2015
EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps	Bio Basic Inc.	BS466	Pladmid preparation
Fast Green FCF	Sigma	F7252-5G	Dye for DNA solution
Fine scissors- sharp	F.S.T.	14060-09	IUE
Fisherbrand Gauze Sponges	Fisher Scientific	1376152	IUE
Gaymar Heating/Cooling	Braintree	TP-700	Heating Pad
Glass pipette puller	Sutter Instrument,	P-97	IUE
Glass pipettes	Sutter Instrument,	BF150-117-10	IUE
Hair Removal Lotion	Nair	N/A	Hair removal
Hartman Hemostats	F.S.T.	13002-10	IUE
Open field maze- homemade acrylic arena	Custom-built	N/A	50 × 50 × 30 cm length-width-height
pCAG-GFP	Addgene	11150	Mammalian expression vector for expression of GFP
Picospritzer III	Parker Hannifin	N/A	pressure injector
Retractor - 2 Pronged Blunt	F.S.T.	17023-13	IUE
Ring forceps	F.S.T.	11103-09	IUE
Sterilizer, dry bead	Sigma	Z378569	sterelize surgical tools
SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY	Havel's	HJ398	Suture muscle
Water bath	Cole-Parmer	EW-12105-84	warming sterile saline