A Rapid In Vivo Bioassay for Developmentally Active Enhancers

Summary

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

Abstract

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS.

In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth.

If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct.

The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

Introduction

Evaluation of systems biology by big data sets (i.e., genome-wide) offers new insights and tool sets for the curation of relevant regulatory molecules and pathways.^1,2 Transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq) generates a genome-wide data set (TF regulatome) of potential regulatory sequences. However, not all of the recovered sites are functionally relevant. The ability to discern signal from noise is a significant limitation of the "omic" data sets.³

With an increasing number of completed genomes available for comparison, one approach is to determine whether a potential regulatory sequence (PRS) is conserved across species and thereby implicate functional relevance. This approach can accelerate discovery in a variety of disciplines, but is particularly helpful in developmental biology where establishing the basic body plan in vertebrate development is controlled by conserved gene sets.⁴ Furthermore, associated regulatory elements are also typically conserved and clustered within introns or associated gene deserts.^5-7 Comparison of the PRS with enhancer-associated marks, such as p300 or H327Ac, are additional tools for the prediction of enhancer activity.^8,9 Although these added screening techniques increase the likelihood that PRSs are functionally relevant, confirmation is still required.

Timmer and coworkers first reported the use of in ovo electroporation as a rapid bioassay for demonstrating enhancer-promoter activity.¹⁰ They injected -PRS linked to reporters into chick neural tubes at Hamburger and Hamilton¹¹ stage (HH) 15 - 17 using various enhancer-promoter combinations and then followed for enhancer activity. Uchikawa generated an inducible reporter construct with a basal herpes simplex thymidine kinase promoter linked to an enhanced GFP reporter (ptk-EGFP).¹² The basal level of activity of this promoter is very low in most tissues allowing for faithful enhancer driven localization of activity. Uchikawa also extended developmental reporting to other tissue involved in early development using a whole embryo electroporation technique at HH 4.¹³ Oberg, Pira and colleagues further extended the capacity of in ovo electroporation to other tissues at later stages of development.^14,15 Thus, the capacity to evaluate enhancer activity in various tissues through a range of developmental stages has been demonstrated.

In this report, the chick as a bioassay is used at multiple stages and sites to determine the activity of PRSs during development. This technique can be used to validate an isolated PRS as the target of a hypothesis driven question or to validate several candidates from a large data set before further analysis.

Protocol

The protocol described below was performed in accordance with Loma Linda University's policy on research involving animals and followed the recommendations within the Federation of Animal Science Societies' (FASS) Guide for the Care and Use of Agricultural Animals in Research and Teaching, 3rd ed. and the National Research Council's Guide for the Care and Use of Laboratory Animals. It is important to check with the local institution regarding their requirements for vertebrate animal use approval when using chick embryos since they are not considered as a vertebrate animal within the FASS guide; however, recommendations for appropriate avian embryo euthanasia are described and adhered to in this lab.

1. Construction of the Reporter Vector

1. Design primers to the potential regulatory sequence (PRS) to contain the restriction endonuclease sequence recognition site for XhoI (CTCGAG) and KpnI (GGTACC). ¹⁶
2. Amplify the region by performing polymerase chain reaction (PCR) using genomic DNA template as previously reported.^17,18
3. Clone the PCR product into the ptk-EGFP expression vector.¹⁹
   1. Use the empty ptk-EGFP expression vector as a negative control to indicate the basal level of expression within a given tissue. Use theβ-actin-driven RFP reporter plasmid as a positive control and an indicator of transfection efficiency.

2. Whole Embryo Electroporation

1. Preparations
   1. Prepare filter paper supports. Punch 4 holes in a cloverleaf pattern onto a 2.2 x 2.2 cm filter paper using a standard hole punch. Sterilize by autoclaving.
   2. Prepare the albumin-agar culture dish.
      1. Dissolve 0.18 g of agar in 30 ml 0.9% sodium chloride on a hotplate, with stirring. Cool in a 42 °C water bath.
      2. Extract the thin albumin by first cracking the narrow end of an unincubated egg and removing the eggshell. Pour out the thick albumin and transfer the thin albumin into a sterile 50 ml conical tube using a large bore plastic transfer pipet. Use enough eggs to get 30 ml of thin albumin (~ 4 eggs). Add 150 µl of 10,000 U/ml penicillin-streptomycin and warm the albumin in a 42 °C water bath.
      3. Combine the albumin and agar and mix by swirling. Transfer 2 ml aliquots to a 35 mm Petri dish, gently swirling the dish to ensure even coverage. Allow the agar to set at room temperature for at least 10 min. Store at 4 °C.
   3. Prepare the cloned PRS vector cocktail: 1 µg/µl of PRS plasmid, 0.3 µg/µl of reporter plasmid, and 0.025% Fast Green FCF stain.
2. Protocol for In Vitro Culturing of Hamburger-Hamilton Stage (HH) 4 Chick Embryos
   1. Incubate fertilized White Leghorn eggs at 39 °C for 18 - 19 hr, according to Hamburger-Hamilton chick staging¹¹.
   2. Spray the surface of the egg with a light mist of 70% ethanol and air-dry.
   3. Crack the egg and empty its content into a 15 cm Petri dish. Carefully remove the thick albumin on the yolk using the blunt end of forceps.
      1. If the embryo is on the side of the yolk, roll the yolk around with the blunt end of forceps until the embryo is in the middle of the yolk.
   4. Place the filter paper on the yolk, with the embryo centered. Cut the vitelline membrane at the edges of the filter paper. Lift the filter paper from the yolk.
      Note: It is important that the vertical orientation of the primitive streak is maintained so that the embryo does not tear.
   5. Place the filter paper on the albumin-agar plate with the hypoblast (yolk side) up. Carefully remove excess yolk with the blunt end of forceps. Gently rinse the embryo with warmed Hank's Balanced Salt Solution (HBSS).
3. Electroporation Protocol
   1. Position the electrodes and pulled glass capillary needle attached to a syringe under a dissecting microscope. Set the 3 mm round platinum cathode at the base of a ~ 2 mm deep chamber under the embryo suspension silicone platform. Mount the 2 mm round platinum anode in a micromanipulator and suspend above the silicone platform. The syringe contains mineral oil as the hydraulic fluid.
   2. Add 100 - 200 µl HBSS into the cathode cavity of the silicone platform. Place the filter paper supporting embryo on the platform with the hypoblast (yolk side) up.
   3. Inject 1 µl PRS vector cocktail between the blastoderm and the vitelline membrane using a pulled glass capillary needle. Follow with a small amount of mineral oil to seal the injection point.
   4. Position the anode about 2 mm above the embryo. Drop ~ 100 µl of HBSS directly over the electrode and embryo. Electroporate with 5 pulses of 5 V for 50 msec at intervals of 100 msec.
      CAUTION: The electroporator can produce high levels of voltage and current. Observe basic lab safety and common sense to prevent injury. This includes not touching the exposed electrodes with bare hands or materials that can conduct electricity while the electroporator is on. Refer to the equipment manual for other safety precautions and potential hazards.
   5. Return the embryo to the agar-albumin plate, with hypoblast up, and incubate in a humidified chamber.
   6. Clean the cathode after each electroporation by aspirating any remaining liquid and yolk. Clean the anode by wiping with a lab tissue.

3. Targeted Regional Electroporation: Neural Tube of HH10 Chicken Embryo 

1. Protocol for Windowing the Egg and Staining the Embryo
   1. Incubate fertilized White Leghorn eggs at 39 °C for 33 - 38 hr, according to Hamburger-Hamilton chick staging¹¹.
   2. Spray a light mist of 70% ethanol onto the eggs and air-dry.
   3. Place the egg on its side on an egg holder. Using a dissecting probe, poke a small hole at the blunt end of the egg. Insert a syringe with an 18 gauge needle through this hole at a downward angle and withdraw 1 - 2 ml of albumin, taking care not to remove any yolk.
   4. Without breaking the underlying shell membrane, poke a shallow hole on the top side of the egg. Carefully remove small portions of the eggshell with the dissecting probe, until ~ 0.5 cm diameter of the underlying membrane is exposed. Break the shell membrane by pushing down on it with the dissecting probe.
   5. Cover the hole with a strip of tape and cut the hole bigger, to ~ 2 cm diameter, using small scissors.
   6. Under a dissecting microscope, identify the blood island surrounding the embryo and add 6 µl of neutral red solution to the middle of this blood island. Wait 2 - 3 min.
   7. Carefully remove the vitelline membrane overlying the embryo using a tungsten needle.
2. Electroporation Protocol
   1. Align the two electrodes in parallel with a fixed inter-electrode distance of 4 mm and mount on a micromanipulator. Position the electrodes on the yolk membrane so that the embryo is centered between the electrodes.
   2. Inject ~ 0.1 µl PRS vector cocktail into the neural tube lumen of the embryo. Add 1 - 2 drops of 1x phosphate buffered saline (PBS) using a transfer pipet. Reposition the electrodes so that they touch the yolk membrane, if necessary.
   3. Apply 5 pulses of 20 V each lasting 50 msec at 950 msec intervals. Add 1 drop of 1x PBS and remove electrodes.
   4. Seal the egg with a piece of transparent tape and return to the egg incubator.
   5. Clean electrodes after each electroporation in bleach, RNase-free water, and 1x PBS to remove residual yolk.

4. Targeted Regional Electroporation: Lateral Plate Mesoderm of HH14 Chicken Embryos

1. Electroporation Protocol
   1. Incubate fertilized White Leghorn eggs at 39 °C for 50 - 53 hr, according to Hamburger-Hamilton chick staging¹¹.
   2. Open and stage the embryos as described above in in Steps 3.1.2 to 3.1.7.
   3. Using a tungsten needle, make a slit onto the yolk membrane outside the periphery of the blood vessel at the tail end of the embryo.
   4. Slide the cathode rod into the yolk through this slit. Position the cathode parallel to and along the length of the embryo. Do not place the cathode directly under the lateral plate mesoderm.
   5. Inject ~ 0.2 µl PRS vector cocktail into the intraembryonic coelom at the level of somite 18, followed by a small amount of mineral oil. The DNA will travel anteriorly and posteriorly along the coelom.
   6. Position the cathode directly under the lateral plate mesoderm, spanning somites 15 - 20. Position the anode above the lateral plate mesoderm with an inter-electrode distance of ~ 2.5 mm. Add 3 - 5 drops of 1x PBS so as to submerge the anode. Adjust the electrodes so that they are not touching the embryo.
   7. Apply 3 pulses of 8 V, 60 msec in duration at 50 msec intervals.Remove cathode from the yolk through the slit.
   8. Add 40 µl 2,500 U/ml of penicillin/ streptomycin. Seal the egg with transparent tape and return to the egg incubator.
   9. Clean electrodes after each electroporation in bleach, RNase-free water, and 1x PBS to remove residual yolk.

5. Fluorescence Analysis

1. Whole Embryo Electroporation
   1. Using forceps, lift the filter paper from the plate and replace the embryo onto the same plate with the hypoblast down. Position the embryo away from residual yolk and cloudy portions of the agar-albumin plate. Alternatively, place the embryo in a new plate for imaging.
   2. Examine the embryos with a fluorescence microscope to visualize transfection efficiency and PRS activity 6 - 48 hr after transfection. To optimize fluorescence intensity, visualize the embryos under high magnification.
2. Targeted Regional Electroporation (TREP)
   1. Carefully dissect the embryo from the egg by cutting the yolk membrane around the embryo with microscissors and transfer to a 35 mm dish containing 1x PBS.
   2. Under a dissecting microscope, remove the extraembryonic membrane with microscissors and forceps.
      1. Keep the microscissors flat and parallel to the embryo to prevent accidentally cutting the embryo. Do not pull or tear the membranes with forceps as the embryo might tear otherwise.
   3. Visualize the embryo in a new 35 mm dish with 1x PBS under bright field and fluorescence microscopy. Use high magnification (40X) to optimize fluorescence intensity.
      1. At high magnification, stitch the images together using an image-editing software to generate the complete image of the targeted tissue.

Discussion

This report describes a bioassay to validate potential regulatory sequences active during development. The approach incorporates the use of a reporter construct, transfection via electroporation and chicken embryos. The process, from sequence determination to validation of activity, can typically be completed within two to four weeks. A critical aspect of this protocol is the efficient transfection of construct into the target tissue. Several steps ensure efficient transfection. The use of endotoxin-free plasmids minimizes post-operative deformation and embryonic death. Cleaning electrodes well after each EP optimizes and standardizes transfection. The voltage and pulses of the electroporator may also need to be adjusted to optimize transfection yet minimize EP related tissue damage. These parameters, once established should be consistently used. For whole embryo EP, removing as much of the yolk from the embryo and filter paper as possible optimizes current flow and keeps the embryo from sagging or tearing from the filter paper. Removing the yolk also makes it easier to visualize the embryo and PRS activity during evaluation.

For targeted regional electroporation (TREP), injection of DNA into a cavity is ideal, since the cavity can retain the concentrated DNA during the time between injection and electroporation. Every attempt should be made to keep this interval short irrespective of the target, but a restricted space is helpful. When an associated cavity or restricted space is not available, a cavity or bubble within the target tissue should be created. For example, Oberg and co-workers created a cavity within limb bud mesoderm by injecting a bolus of DNA followed by a bubble of mineral oil.¹⁴ Retention of DNA by this approach promoted efficient transfection of limb bud mesoderm. If direct tissue contact by the electrodes is necessary to direct current to the target tissue, insulate the electrodes as described by Oberg and co-workers to minimize short circuits that can reduce transfection efficiency.¹⁴ Be aware that direct contact will also cause some local tissue damage. If TREP of an organ has yet to be described, the approach should be optimized first with the RFP construct to ensure effective transfection of the targeted tissue with minimal tissue damage. The major limitation of this approach is the transfection of organs that form in more advances stages of development or are physically difficult to target within the embryo such as the metanephric kidney.

A number of in vitro and in vivo approaches have been used to validate potential regulatory sequences. Validation of an enhancer element can be accomplished in a relevant in vitro cell line.²¹ Although this approach can be useful, cell lines are immortal and out of context from their original derivation and thus, may not represent in vivo activity. Even primary cells grown in culture are out of context to their in situ relationships and rapidly lose some of their differentiated features.^22,23 For a transcription factor that has precise spatial and temporal expression during development, context is critical for functional validation. Analysis of enhancer activity in transgenic mice follows a protocol similar to what is described in this report with generation of a reporter construct, typically using the LacZ reporter, although GFP has also been described.^24,25 The mouse transgenic approach requires the injection of the construct into mouse embryonic stem cells, typically injecting 100 - 300 cells for recovery 3 - 5 embryos, 3 - 7 week after construct generation, and costs of around $ 2,500 per construct. Furthermore, since evaluation occurs post-harvesting only one developmental stage can be examined per embryo. Thus, using the mouse transgenic approach to evaluate developmentally active vertebrate enhancers is limited by embryonic stage, cost, time and efficiency. The zebrafish model is a more assessable and accessible model for enhancer activity but is also a vertebrate model that is more divergent from human than either mouse or chicken.⁷ Thus, for the analysis of limb development related enhancers, the chick or mouse models would be more informative. The zebrafish model also requires establishing a fish facility to breed and maintain a colony, which is not a trivial undertaking.

The chick is a common well-studied vertebrate developmental model for several major organ systems including brain, heart and limbs. Fertile eggs are readily available and require only an avian incubator for development. Similar to the zebrafish model, assessment of enhancer activity in chick embryos is rapid, within 6 hr after transfection, and as in zebrafish, enhancer activity can be followed through multiple stages of development. The most expensive item required to setup up a chick bioassay system for enhancer activity in most labs is the electroporation unit that is presently around $ 14 k, which can be used for hundreds of transfections and bioassays of potential regulatory sequences. Considering the cost of mouse transgenics or establishing a fish facility, the chick bioassay is quite cost effective. Overall, the chick bioassay as described provides a rapid system for validating enhancers active during vertebrate development. The approach can be used to supplement high throughput techniques to confirm functional relevance of data sets. Alternatively, this approach can be used to validate targets extracted from genomic data sets that are to be used in hypothesis-driven, mechanistic investigations.

Materials

Name	Company	Catalog Number	Comments
Bacto-Agar	BD Biosciences	204010
Sodium chloride	Sigma	S9888
Hank's Balanced Salt Solution (HBSS)	Sigma	H6136	Hank's Balanced Salt Solution (HBSS), Without sodium bicarbonate
Penicillin-streptomycin	Life Technologies	15140-122
Ethanol 200 proof			dilute to 70%
Endofree Maxiprep Plasmid Kit	Qiagen	12362	EndoFree Plasmid Maxi Kit
White Leghorn fertilized eggs	Chino Valley Ranchers
Whatman Grade 4 filter paper	GE Healthcare Life Sciences	1004-240	filter paper support (a range of grades are ssufficent to provide stability during growth)
50ml conical tube	VWR	21008-242
35mm petri dish	VWR	627161	Sterile Petri Dishes, 35W x 10H mm
15cm petri dish	VWR	82050-600	Sterile Petri Dishes, 145W x 20H mm
Transfer pipet	VWR	414004-042
Capillary tube	World Precision Instruments	1B100F-6
Terasaki dish	VWR	82050-706	dish from which to aspirate DNA
mineral oil
Standard hole punch
Hotplate  stirrer
Stir bar
Water bath
Round Platinum 3mm Petri dish electrode	Protech International Inc	CUY700-P3E	electrode for whole embryo electroporation
Round Platinum 2mm cover electrode	Protech International Inc	CUY700-P2L	electrode for whole embryo electroporation
Humidified chamber
Dissecting microscope
Dissecting fluorescence microscope
Forceps			`
Standard surgical scissors
CUY21-EDIT Square Wave Electroporator	Protech International	CUY21-EDIT	electroporator
Micropipette	Hamilton Company
Micromanipulator
Egg holder (nest)			modeling clay  shaped on a 10cm petri dish
Dissecting probe			to window the egg
Parallel Fixed Needle Electrode	Protech International Inc	CUY610-P4.4	4mm Gap & Platinum 4mm Tip, electrode for electroporation of neural tube
Tungsten wire	Omega Engineering, Inc	W5W26-010	Tungsten 5% Re vs. Tungsten 26% Re, 0.010, unsheathed, used to make fine cuts on embryo
Needle holder
Platinum Z-Shape Blunt Needle Electrode	Protech International Inc	CUY611-P7.4	7mm Length x 4mm Exposed Tip, electrode for electroporation of lateral plate mesoderm
18 gauge needle
10cc syringe
Scotch transparent tape	Office Depot	305324
Neutral red solution 0.25%	Fisher Scientific	N12925	to stain the embryo
Dulbecco’s Phosphate Buffered Saline	Sigma aldrich	D5773	Dulbecco’s Phosphate Buffered Saline, Without sodium bicarbonate
Household bleach			for electrode cleaning
Fast green FGF (0.25%)	Specrtum Chemical Mfg Corp	FA105
Predicted regulatory region Plasmid
Reporter pasmid	Several options		The ptkEGFP was a kind gift from Uchikawa and Kondoh, but Adgene has a ptkRFP that could be used for the reporter and pCAG-GFP for the transfection control.
TE buffer	Qiagen	included in Endofree Maxiprep Plasmid Kit	for dilution of DNA