AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Summary

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

Abstract

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

Introduction

Enhancers are genomic cis-regulatory elements that serve as transcription factor binding sites and can drive the expression of a target gene in a spatiotemporally specific manner^1,2. They are differentially active in different cell types, tissues, and stages of development and can be substrates of disease risk-related genomic variation^3,4. Thus, the need to understand the dynamics of enhancer function is critical to progress in both translational and basic science applications within genomics. In silico predictions of enhancer activity can ser....

Protocol

This protocol has been approved by the UC Davis Institutional Animal Care and Use Committee (Protocol #22339) and the UC Davis Institutional Biosafety Committee (BUA-R1903). This protocol has been tested on C57BL/6J mice of both sexes at postnatal day 0-1.

1. Clone the enhancer candidate sequence into the AAV vector plasmid.

NOTE: The representative protocols are given, but the cloning strategy has a high degree of flexibility.

1. Select or.......

Discussion

This protocol describes an rAAV-based method for the deployment of enhancer-driven transgenes in the postnatal mouse brain. In this generalized protocol, a candidate enhancer, a minimal promoter, a reporter gene, and an optional barcode sequence are cloned into an AAV plasmid backbone. These experiments can be done with a single candidate enhancer sequence or with many sequences in parallel. The plasmid is packaged into an rAAV and delivered to the postnatal mouse brain. After a period of time to allow for virus tra.......

Materials

Name	Company	Catalog Number	Comments
10x Citrate Buffer	Sigma-Aldrich	C9999-1000ML
5'-gatcactctcggcatggac-3'	Integrated DNA Technologies	N/A: Custom designed	Forward primer for verifying clones after transformation. These primers are specific to the vector used and were designed for the specific vector used in our experiments.
5'-gatggctggcaactagaagg-3'	Integrated DNA Technologies	N/A: Custom designed	Reverse primer for verifying clones after transformation. These primers are specific to the vector used and were designed for the specific vector used in our experiments.
Agarose	VWR	VWRVN605-500G
Aspirator tube assemblies	Sigma-Aldrich	A5177-5EA	for mouth-driven delivery of rAAV
Bacteriological petri dishes	Thermo Fisher Scientific	08-757-100D
Carbenicillin	Sigma-Aldrich	C1389-5G
Chicken IgY anti-GFP	Thermo Fisher Scientific	A10262
Confocal microscope	Zeiss	LSM900	The images were taken on the LSM800 model, but Zeiss launched the LSM900 model in recent years to replace LSM800.
Conical centrifuge tubes 15 mL	Thermo Fisher Scientific	12-565-269
Cryomolds	Thermo Fisher Scientific	NC9806558	These molds are suitable for P28 mouse brain. Other sizes may be more suitable for larger or smaller tissues.
DAPI	Sigma-Aldrich	D9542-10MG
Dissecting scissors, 4.5"	VWR	82027-578
Donkey anti-chicken AlexaFlour-488	Jackson ImmunoResearch	703-545-155
Dulbecco's PBS 1x	Thermo Fisher Scientific	MT21031CV
Eppendorf Microcentrifuge tubes 2.0 mL	Thermo Fisher Scientific	22431048
Falcon round-bottom tubes 14 mL	Thermo Fisher Scientific	352059
Fast Green dye	Grainger	F0099-1G
Fine detail paint brush set	Artbrush Tower	B014GWCLFO
Gibson Assembly Master Mix	NEB	E2611S
Glass capillary tubes	Drummond Scientific Company	5-000-2005
HiSpeed Plasmid Maxi Kit	QIAGEN	12663	Commercial plasmid maxi prep kit
HyClone HyPure Molecular Biology Grade Water	VWR	SH30538.03
IV butterfly infusion set with 12" tubing and 25G needle	Thermo Fisher Scientific	26708
Kimwipes	Kimberly Clark	34155	Lint-free wipe
LB Agar	Thermo Fisher Scientific	BP1425-500	LB agar pre-mix for selective media
McPherson Vannas iris scissor	Integra LifeSciences	360-215
Mineral oil	Sigma Life Science	69794-500ML
NEB Stable Competent E. coli	NEB	C3040I
NucleoSpin Gel and PCR Clean-Up	Takara	740609.5	Kit for enzymatic reaction cleanup and gel extraction
OCT medium	VWR	25608-930
Orbital shaker	Cole Parmer	60-100
Paraformaldehyde	Sigma-Aldrich	158127-500G
PCR strip tubes 0.2 mL	VWR	490003-692
Peristaltic pump	Gilson	F155005
Phosphate buffered saline (PBS) 10x	Thermo Fisher Scientific	70011044
Phusion Hot Start II High Fidelity DNA Polymerase	Thermo Fisher Scientific	F549L
Powdered milk	Sunny Select
ProLong Gold Antifade Mountant	Thermo Fisher Scientific	P36934
QIAquick PCR Purification Kit	QIAGEN	28106
rCutSmart Buffer	NEB	B6004S	Buffer for restriction digest with PacI, AscI, and XmaI
Restriction enzyme: AscI	NEB	R0558L
Restriction enzyme: PacI	NEB	R0547L
Restriction enzyme: XmaI	NEB	R0180L
SOC outgrowth medium	NEB	B0920S	Recovery medium after transformation
Sucrose (RNase/DNase free)	Millipore Sigma	033522.5KG
TAE buffer	Apex	20-194
Transfer tubing	Gilson	F1179941	For peristaltic pump
Triton X100	Sigma-Aldrich	X100-100ML
Wizard Plus SV Minipreps DNA Purification System	Thermo Fisher Scientific	A1460	Plasmid mini prep kit