A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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 manner1,2. They are differentially active in different cell types, tissues, and stages of development and can be substrates of disease risk-related genomic variation3,4. 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 serve as excellent resources for generating hypotheses as to enhancer capability5,6. Such predicted enhancer activity can require additional validation and interrogation for a full understanding of the functional activity. Enhancer reporter assays have proved valuable for this purpose across a variety of systems, from cells to animals7,8,9. Towards extending these studies in a flexible and cost-effective transient in vivo context, this protocol describes the use of in vivo AAV-based methods to test putative enhancer sequences for their ability to drive the expression of an ectopic reporter gene in the postnatal mouse brain. This family of methods has utility for interrogating single candidate sequences or parallel library screening and is relevant for basic and translational research.
This method combines in a single plasmid a putative enhancer candidate DNA sequence with a reporter gene (here EGFP), under the control of a minimal promoter that alone does not drive significant expression. The plasmid is packaged into recombinant AAV (rAAV) and injected into an animal model. While the application here is to the brain, various rAAV serotypes enable infection across different tissue types so that this approach can be extended to other systems10. After a period of time, the brain can be collected and assayed for the expression of the reporter gene. Strong expression, compared with controls, indicates that the tested candidate sequence was able to "enhance" the expression of the gene (Figure 1). This simple design offers an easy and clear approach to test a sequence for enhancer activity in vivo in the brain.
In addition to testing for enhancer capability of a sequence, this method can be combined with techniques to determine cell-type enhancer activity. In sequence-based approaches to determining differential enhancer activity, sorting cells on cell-type-specific markers prior to DNA and RNA sequencing can allow researchers to determine if different cell types show differential enhancer activity, as was described in Gisselbrecht et al.11. In imaging-based approaches, co-labeling images with cell-type-specific markers allows examination of whether cells exhibiting enhancer-driven fluorescence also display cell-type markers of interest12,13,14,15,16. Enhancer reporter assays enable direct testing of risk-associated allelic variation in enhancers for effects on enhancer capability. The vast majority of risk loci identified in genome-wide association studies (GWAS) lie in non-coding regions of the genome17. Functional annotation studies of these risk loci indicate that a large portion likely act as enhancers18,19,20. MPRA deployment in vivo can allow testing of these risk-associated variants for enhancer activity in brain12,21. Finally, delivery and collection at different time points can offer insights into the developmental stages during which an enhancer is active.
Enhancer-reporter plasmid designs are diverse and can be customized to suit experimental goals. There are several options for minimal promoters that have been used in enhancer research, such as the human β-globin minimal promoter22 and the mouse Hsp68 minimal promoter23. These promoters are known to drive low levels of expression unless coupled with an enhancer element to activate them. In contrast, constitutive promoter elements drive strong expression of the transgene, useful for positive control or to test for enhancer function against a background of robust expression. Common choices for constitutive promoters include CAG, a hybrid promoter derived from the chicken β-actin promoter and the cytomegalovirus immediate-early enhancer24, or human EF1α25. Since enhancers are known to work bidirectionally26, the orientation and location of the enhancer relative to the minimal promoter are flexible (Figure 2A). Traditional enhancer-reporter assays place the enhancer upstream of the promoter and, in library deliveries, include a barcode sequence downstream of the reporter gene to associate sequencing reads with the tested enhancer27. However, enhancers can also be placed in the open reading frame of the reporter gene and serve as their own barcode sequence, as is done in STARR-seq28. The protocol described here utilizes the STARR-seq assay design, placing the candidate enhancer sequence into the 3' UTR of the reporter gene. While the STARR-seq orientation offers the benefit of more streamlined cloning, it is less well understood than the conventional approach and may induce variable RNA stability between constructs. The described methods can be easily adapted to either the STARR-seq or conventional orientation with minor alterations to the cloning process that have been described elsewhere27,29.
Different methods of AAV delivery can be employed to further customize this technique to fit experimental goals (Figure 2B). Direct intracranial injections, described further in this protocol, deliver a high concentration of virus directly to the brain30. This gives a high transduction efficiency centered at the site of injection, making this an excellent technique for experiments looking to maximize the density of transduced cells over an area of tissue. Stereotactic injection can help standardize the site of injection across animals for reproducible localized transduction. Intracranial injections are most straightforward in early postnatal animals. As an alternative technique, systemic injections can deliver transgenes using AAVs with serotypes capable of crossing the blood-brain barrier31. Tail-vein injections allow the virus to circulate throughout the body, enabling generalized delivery across many tissues10. Retro-orbital injections are another systemic injection technique that delivers the virus behind the eye into the retro-orbital sinus32. This offers a more direct route for the AAV from the venous system to the brain, resulting in a higher concentration of transduced cells in the brain than injections into more peripheral blood vessels33.
Another flexible aspect of this technique is the method of readout. Broadly, options can be described as reporter-based or sequencing-based (Figure 2C). Incorporating a fluorescent reporter such as GFP into the open reading frame of the construct results in the expression of the fluorescent protein in any transduced cells where the candidate enhancer drove expression. Labeling and imaging techniques such as immunohistochemistry enable signal amplification. Sequencing-based readout techniques involve identifying sequences from the delivered construct in RNA collected from the tissue. By quantifying the amount of viral DNA that was initially delivered, the comparison of expressed RNA versus delivered DNA can be used to determine the degree to which a tested enhancer sequence was capable of driving increased expression of the transgene, for example, in the context of a massively parallel reporter assay (MPRA). MPRAs offer a powerful expansion of these techniques to test up to thousands of candidate enhancers for activity simultaneously and have been described extensively in genomics research12,27,34,35,36. Higher throughput screening is achieved by executing cloning, packaging, delivery, and sequencing steps for candidate enhancers in batch rather than individually.
Candidate enhancer selection provides another opportunity for flexibility (Figure 2D). For example, this assay can be used to identify enhancers of a specific gene, to ascertain the function of non-coding DNA regions of interest, or to determine specific cell types or developmental stages during which an enhancer is active - all of which serve goals both in basic science and in disease research. Generally, candidate enhancer selection is driven by in silico predictions of enhancer activity. Commonly, in silico predictions include ChIP-seq for histone modifications that indicate likely enhancers, such as H3K27ac37 and chromatin accessibility mapping38. Finally, a growing area of research is the function-based screening of synthetically designed enhancer elements, enabling studies of how enhancer sequence directs function39 and the design of enhancers with specific properties40.
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.
2. Obtain packaged rAAV.
3. Intracranially inject rAAV-packaged plasmid into neonatal mice.
4. Collect tissue and perform immunohistochemistry.
5. Image and analyze brain tissue sections for enhancer activity.
Using these methods, a 915 bp sequence in the psychiatric risk-associated third intron of the gene CACNA1C19,49,50 was tested for enhancer activity in the postnatal mouse brain. This sequence was discovered in an MPRA of 345 candidate enhancer sequences centered on psychiatric and neurological risk SNPs12 and characterization experiments are described here as a general example. C57BL/6 mice ...
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...
The authors declare no competing financial interests.
Sequencing was performed at the UC Davis DNA Technologies Core. We thank the lab of Lin Tian at UC Davis for training on rAAV packaging and generously gifting us AAV helper and rep/cap plasmids. This work was supported by NIH/NIGMS R35GM119831.
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 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved