Mouse In Vivo Placental Targeted CRISPR Manipulation

Podsumowanie

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Streszczenie

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Wprowadzenie

The placenta is an essential organ involved in the development of the fetus. The main role of the placenta is to provide essential factors and regulate the transfer of nutrients and waste to and from the fetus. Mammalian placentas are composed of both fetal and maternal tissue, which make up the fetal-maternal interface, and, thus, the genetics of both the mother and fetus impact function¹. Genetic anomalies or impaired function of the placenta can drastically alter fetal development. Previous work has shown that placental genetics and development are associated with the altered development of specific organ systems in the fetus. Particularly, abnormalities in the placenta are linked with changes in the fetal brain, heart, and vascular system^2,3,4,5.

The transport of hormones, growth factors, and other molecules from the placenta to the fetus plays a major role in fetal development⁶. It has been shown that altering the placental production of specific molecules can alter neurodevelopment. Maternal inflammation can increase the production of serotonin by altering tryptophan (TRP) metabolic gene expression in the placenta, which subsequently creates an accumulation of serotonin in the fetal brain⁷. Other studies have found placental abnormalities alongside heart defects. Abnormalities in the placenta are thought to contribute to congenital heart defects, the most common birth defect in humans⁸. A recent study has identified several genes that have similar cellular pathways in both the placenta and heart. If disrupted, these pathways could cause defects in both organs⁹. The defects in the placenta may exacerbate congenital heart defects. The role of placental genetics and function on specific fetal organ system development is an emerging field of study.

Mice have hemochorial placentas and other features of human placentas, which makes them highly useful models for studying human disease¹. Despite the importance of the placenta, there is currently a lack of targeted in vivo genetic manipulations. Furthermore, there are currently more options available for knockouts or knockdowns than overexpression or gain-of-function manipulations in the placenta¹⁰. There are several transgenic Cre-expressing lines for placental-specific manipulation, each in different trophoblast lineages at different time points. These include Cyp19-Cre, Ada/Tpbpa-Cre, PDGFRα-CreER, and Gcm1-Cre^11,12,13,14. While these Cre transgenes are efficient, they may not be capable of manipulating some genes at specific time points. Another commonly used method to either knockout or overexpress placental gene expression is the insertion of lentiviral vectors into blastocyst culture, which causes a trophoblast-specific genetic manipulation^15,16. This technique allows for a robust change in the placental gene expression early in development. The use of RNA interference in vivo has been sparsely utilized in the placenta. The insertion of shRNA plasmids can be performed similarly to the CRIPSR technique described in this paper. This has been done at E13.5 to successfully decrease PlGF expression in the placenta, with impacts on offspring brain vasculature¹⁷.

In addition to techniques that are primarily used for knockout or knockdown, inducing overexpression is commonly performed with adenoviruses or the insertion of an exogenous protein. The techniques used for overexpression have varying rates of success and have mostly been performed later in gestation. To investigate the role of insulin-like growth factor 1 (IGF-1) in placental function, an adenoviral-mediated placental gene transfer was performed to induce the overexpression of the IGF-1 gene^18,19. This was performed late in mouse gestation on E18.5 via direct placental injection. To provide additional options and circumvent possible failures of established placental genetic manipulations, such as Cre-Lox combination failures, the possible toxicity of adenoviruses, and the off-target effects of shRNA, in vivo direct CRISPR manipulation of the placenta can be used^20,21,22. This model was developed to address the lack of overexpression models and to create a model with flexibility.

This technique is based upon the work of Lecuyer et al., in which shRNA and CRISPR plasmids were targeted directly in vivo to mouse placentas to alter PlGF expression¹⁷. This technique can be used to directly alter placental gene expression using CRISPR manipulation at multiple time points; for this work, E12.5 was selected. The placenta has matured by this point and is large enough to manipulate, allowing for the insertion of a specific CRISPR plasmid on E12.5, which can have a significant impact on fetal development from mid to late pregnancy^23,24. Unlike transgenic approaches, but similar to viral inductions or RNA interference, this technique allows for overexpression or knockout at particular time points using a relatively advanced surgical approach, thus avoiding possible impaired placentation or embryonic lethality from earlier changes. As only a few placentas receive the experimental or control plasmid within a litter, the approach allows for two types of internal controls. These controls are those injected and electroporated with the appropriate control plasmid and those that receive no direct manipulation. This technique was optimized to create an overexpression of the IGF-1 gene in the mouse placenta via a synergistic activation mediator (SAM) CRISPR plasmid. The IGF-1 gene was chosen, as IGF-1 is an essential growth hormone delivered to the fetus that is primarily produced in the placenta prior to birth^25,26. This new placental-targeted CRISPR technique will allow for direct manipulation to help define the connection between placental function and fetal development.

Protokół

All procedures were performed in accordance and compliance with federal regulations and University of Iowa policy and were approved by Institutional Animal Care and Use Committee.

1. Animals and husbandry

1. Keep the animals in a 12 h daylight cycle with food and water ad libitum.
2. Use CD-1 female mice aged 8-15 weeks. Use the presence of a copulatory plug to identify E0.5.
3. On E0.5, singly house the pregnant dams.

2. Calibration of the micropipette

NOTE: The calibration of the micropipette should be performed prior to surgery when possible.

1. Prior to making and calibrating the micropipette, dilute all the plasmids to the recommended concentration (0.1 µg/µL) in DNase-free water. Mix the plasmid with appropriately diluted Fast Green dye (1 µg/µL in PBS) (final plasmid concentration: 0.06 µg/µL).
2. Pull micropipettes using 10 cm glass capillaries with an outer diameter of 1.5 mm and an inner diameter of 0.86 mm with a micropipette puller.
3. Carefully break off the tip of a micropipette (2-3 mm) with sterile forceps.
4. Load the micropipette into a microcapillary tip attached to a microinjector. Ensure the microinjector is attached to the microinjection machine and that there are sufficient levels of nitrogen to calibrate the micropipette.
5. Once the micropipette is attached to the microinjector, load it with the Fast Green dye solution to perform the calibration (1 µg/µL in PBS). Calibrate the micropipette to inject a volume of approximately 3.5 µL. Empty ("clear") the micropipette in preparation for loading the plasmid solutions as below.
   ​NOTE: Each micropipette will be slightly different. To avoid damaging the placenta during the injection, the injection time should be set between 0.5-1.5 s. The pressure should be set between 1-8.5 psi. Calibrate each micropipette separately; if the micropipette cannot be calibrated within these parameters, then it should not be used.

3. Surgery (Figure 1A)

NOTE: To prepare, clean the surfaces of both the preparation and surgical areas with 70% ethanol. Place an absorbent underpad in the preparation area. In the surgery area, place a heating pad down, and then place an absorbent underpad on top of this. Sterilize all the tools prior to surgery. The time the dam is under anesthesia should be under 1 h.

1. Anesthetization
   1. Administer 5 mg/kg of NSAID (meloxicam) or another approved analgesic to the pregnant dam 30 min to 1 h before surgery.
   2. Place the pregnant dam in an induction chamber attached to an isoflurane vaporizer.
   3. Set the vaporizer to 4% isoflurane and 3.5 L/min oxygen.
   4. Once anesthetization has been confirmed by the lack of response to a toe pinch and a reduced breathing rate, remove the dam from the induction chamber to the preparation area.
2. Surgery preparation
   1. In the preparation area, place the dam supine with a nose cone.
   2. Reduce the vaporizer to 2% isoflurane and 3.5 L/min oxygen while the dam is in the nose cone.
   3. Shave the abdomen of the dam thoroughly and remove excess fur. Alternate coating the shaved abdomen with povidone solution and 70% ethanol three times using sterile cotton-tipped applicators. Apply final coat of povidone solution. To prevent corneal drying, place artificial tear gel over both eyes of the dam (Figure 2A, B).
   4. After preparation, move the dam with the nose cone to the designated surgery area.
3. Uterine laparotomy
   NOTE: Use sterile gloves throughout the surgical procedure. Change the gloves if any non-sterile surface is contacted. 
   1. In the surgery area, place the dam supine, and secure the nose cone in place with tape. Set the heating pad underneath the absorbent pad to 45 °C.
   2. Using forceps and scissors, make an approximate 2 cm midline incision through the skin. Use forceps to tent the skin, and make a vertical incision into the skin. After this, make another similarly sized incision through the peritoneum to expose the uterine horns. Using forceps, tent the peritoneum while making the vertical incision (Figure 2C, D).
      NOTE: Failure to properly tent while incising the peritoneum may lead to a fatal incision of the intestines.
   3. Gently massage the uterine horns through the incision by pressing on the sides of the abdomen. Do this by carefully guiding the uterus without tools, using only fingers to avoid accidental injury. Place the exposed uterus on top of a sterile surgical drape covering the abdomen of the dam and keep it moist throughout surgery with drops of sterile saline, which can be warmed to 30 °C prior to use as needed (Figure 2E, F).
      NOTE: The uterine horns can be identified as described in Wang et al.²⁷.
   4. Once the uterus is exposed, select three pairs of placentas for manipulation.
      NOTE: The placentas can be identified as described by Elmore et al.²⁴. To maximize the survival of the embryos, no more than 6 placentas should be treated. If there are fewer than 12 embryos present, no more than 4 placentas should be injected. Select two adjacent placentas so that one receives a control injection and the other receives the experimental plasmid. The use of two adjacent placentas allows for a better comparison of placentas in similar locations in the uterus and also enables an increased rate of survival. The selected placental pairs are chosen randomly and spaced throughout both uterine horns (Figure 1B).
   5. Record the location of placental manipulations and organization of the embryos within the uterine horns so that the embryos and placentas can be identified during collection, as one dam will carry both control and experimentally treated placentas/embryos.
4. Placental injection and electroporation of the control plasmid
   NOTE: To maintain an aseptic technique, sterilize the electroporation paddles and microinjector with a cold sterilant before use. Change gloves if any non-sterile surface is contacted. 
   1. Using the calibrated micropipette, load a sufficient quantity of the appropriate control plasmid for three injections. Perform all the injections at a depth of ~0.5 mm laterally into the placenta between the decidua (white) and junctional zone (dark red) (Figure 3A-F).
   2. Perform injections in the three control placentas.
      NOTE: Perform all the control plasmid injections prior to the experimental injections to avoid cross-contamination of the plasmids with the micropipette. This will allow for the same micropipette to be used, as changing micropipettes and calibration time drastically increases the surgery time and decreases dam survival.
   3. Perform electroporation of the control plasmid-injected placentas within 2 min of the injection.
   4. For electroporation, use a pair of 3 mm paddles attached to an electroporation machine. To ensure CRISPR incorporation efficiency and the viability of embryos, use the following electroporation settings: 2 pulses, 30 V, 30 ms pulse, 970 ms pulse off, unipolar.
   5. After injection but immediately prior to electroporation, coat the places of contact with sterile saline, applying the saline precisely to the three sites on the uterine wall and the paddles with a dropper or syringe.
   6. Gently press the electroporation paddles on the lateral sides of the placenta. Place the anode paddle over the injection site and the cathode directly opposite (Figure 4A-C).
   7. Press the pulse on the electroporation machine, and wait for the two pulses to complete prior to removing the electroporation paddles.
      NOTE: A small amount of white foam is often seen between the paddles and placenta during pulses. If this does not occur, check the voltage from the paddles with a voltmeter before further use. If the reading on the voltmeter does not match the electroporation voltage setting, the paddles are nonfunctional.
5. Placental injection and electroporation of the experimental plasmid
   1. Follow the same instructions from step 3.4.1. to step 3.4.2. using the experimental plasmid instead of the control plasmid.
   2. Perform electroporation of all three experimental injected placentas within 2 min of the injection. This should be performed in the same manner as for those injected with the control plasmids. Follow step 3.4.4. to step 3.4.7.
6. Completion of the surgery
   1. Once the placental manipulation is complete, gently massage the uterine horns back inside the abdominal cavity using only fingers (Figure 5A).
   2. First, perform double-knotted single sutures on the peritoneum layer using coated and braided dissolvable sutures that are 45 cm long with a 13 mm 3/8c needle alloy. Space the sutures 2-3 mm apart (Figure 5B).
   3. After suturing the peritoneum layer, suture the skin with dissolvable sutures. Triple knot the single sutures 2-3 mm apart to ensure that the dam cannot undo the suturing (Figure 5C).
   4. Once the suturing is complete, set the isoflurane to 1% and the oxygen to 3.5 L/min, and apply tissue adhesive to the sutures on the skin (Figure 5D).
      NOTE: Tissue adhesive is optional but recommended to prevent re-opening of the incision due to dam chewing.
   5. When the tissue adhesive has dried, turn off the vaporizer, and remove the dam from the surgery area. Place the dam in a supportive cage on its back.

4. Post-surgery care and monitoring

1. Allow the pregnant dam to recuperate in a clean cage under supervision for a minimum of 30 min to ensure no immediate complications of the surgery. Observe until it is fully ambulatory and can flip onto its feet without assistance. Singly house the dam post-surgery.
2. Follow the institutional post-operative care and monitoring until embryo collection. Record the dam weight, and monitor the sutures and the incision site daily.

5. E14.5 placental collection

1. On E14.5, deeply anesthetize the dam with a ketamine/xylazine cocktail (1 mg/mL ketamine and 0.1 mg/mL xylazine), and then cervically dislocate the dam.
2. Make a V-shaped incision into the abdomen of the dam with scissors, and remove the uterus. Place immediately onto a 5 cm Petri dish on ice. Using forceps, carefully remove the embryo and the corresponding placenta from the uterus.
   NOTE: Keep a record of the embryo location and corresponding placenta in the uterine horns to determine which received the direct manipulation during surgery.
3. Record the placental weights. Using RNAse-free forceps and razors, cut the placenta in half down the midline. Put one half into 4% PFA at 4 °C. Cut the remaining half in half again, and store the remaining two quarters at −80 °C in two tubes, one with RNA storage reagent.
   ​NOTE: Embryos and other maternal tissues can be stored from the collection at −80 °C for future use.

6. Placental gene expression analysis

1. Use the quarter of the placenta stored at −80 °C in RNA storage reagent.
2. Process the placentas for qPCR as described in Elser et al. using the Trizol method for RNA isolation, a spectrophotometer for RNA concentration, a cDNA synthesis kit, and qPCR with SYBR Green Master Mix²⁸. In place of the Turbo DNAfree kit DNAse referenced in Elser et al., use an RNAcleanup kit after the Trizol RNA isolation to ensure the samples do not contain contaminants²⁸.
   NOTE: Read the material safety data sheet (MSDS) for Trizol, and use it in a chemical fume hood.
3. Assess the plasmid insertion of the control plasmid with GFP primers and of the experimental plasmid with BLAST primers (primers listed in Supplementary Table 1). Use the CT value to determine the presence of the plasmid.
   NOTE: CT values above 35 are false positives, and only those below 35 should be considered a positive indicator that the plasmid was successfully inserted.
4. Assess IGF-1 placental expression normalized to the housekeeping gene 18s (primers listed in Supplementary Table 1). Use the ddCT method to calculate the fold change, and then calculate the normalized fold change of the experimental samples to the mean fold change of the control samples.

7. Placental protein level analysis

1. Use the quarter of the placenta that has been stored at −80 °C. Homogenize the tissue in a buffer solution made of 11.5 mM Tris HCl, 5 mM MgCl₂, and 10 mM protease inhibitor in diH₂O with a final pH of 7.4. Use a handheld homogenizer and pestle to break up the tissue.
   NOTE: The sample should not exceed 10% of the volume of the buffer.
2. Dilute the homogenized samples in the buffer at 1:12, so that they are within the detectable range of a bicinchoninic acid assay (BCA) kit. Perform the BCA assay according to the manufacturer's instructions, and quantify the total protein using a plate reader.
3. After performing the BCA assay, normalize all the samples to the same total protein concentration of 2 mg/mL for the IGF-1 ELISA, as done in Gumusoglu et al.²⁹.
4. Perform the IGF-1 ELISA according to the manufacturer's instructions, and quantify the IGF-1 protein levels with a plate reader using a standard concentration curve.

8. Spatial CRIPSR verification using fluorescent in situ hybridization labeling

1. After the placental halves have been appropriately fixed in 4% PFA at 4 °C for 1-3 days, move them into 20% sucrose at 4 °C before freezing them in optimal cutting temperature (OCT) compound.
2. Serially section the OCT-embedded placenta halves in a −20 °C cryostat into 10 µm sections, and place them onto slides to be labeled. Section the placenta halves so that all three subregions are visible. Store the slides at −80 °C until use for in situ hybridization.
3. Perform fluorescence in situ hybridization (FISH) labeling following the manufacturer's protocols. Hybridize one slide with a dCas9-3xNLS-VP64 probe and a second "sister" slide of the same placenta with a Prl8a8 probe.
   NOTE: The dCas9-3xNLS-VP64 probe detects the presence of the overexpression plasmid. The Prl8a8 probe highlights spongiotrophoblasts of the junctional zone, which allows for the subregions of the placenta to be identifiable. These two probes are labeled on separate "sister" slides to avoid interference of multi-channel fluorescence with the green autofluorescence in the placentas.
4. Label both probes with the detection dye (Opal 620). After completing the manufacturer's protocol for FISH, apply DAPI mounting medium, and place a coverslip on the slide. Seal the coverslip with clear nail polish.
5. Image the slides on an upright compound fluorescence microscope, and process them using an appropriate image processing software. Here, the CellSens software was used.

Wyniki

General procedure outcomes (Figure 6)
In the study, there were three manipulated groups. These included placentas injected with a general CRISPR Cas9 control plasmid (Cas9 Control), an activation control CRISPR plasmid (Act Control), or an IGF-1 SAM activation plasmid (Igf1-OE). The Cas9 Control is better suited for knockout plasmids, and the activation control is better suited for overexpression/activation plasmids. To assess the viability ch...

Dyskusje

The placenta is a primary regulator of fetal growth, and as previously noted, changes in placental gene expression or function may significantly impact fetal development⁶. The protocol outlined here can be used to perform a targeted in vivo CRISPR manipulation of the mouse placenta using a relatively advanced surgical approach. This technique allows for a significant yield of viable embryos and their corresponding placentas that can be used for further study (Figure 6...

Materiały

Name	Company	Catalog Number	Comments
1.5 ml Tubes	USA Scientific Inc	1615-5500
4% Paraformeldhyde (PFA) in PBS	Thermo Fisher Scientific	J61899.AP
96 Well plate	Cornings	3598	For BCA kit
Absorbent Underpads	Fisher Scientific	14-206-62
Activation Control Plasmid	Santa Cruz Biotechnology	sc-437275	Dnase-free water provided for dilution
AMV Reverse Transcriptase	New England Biolabs	M0277L	Use for cDNA synthesis
Anesthetic Gas Vaporizor	Vetamac	VAD-601TT	VAD-compact vaporizer
Artifical Tear Gel	Akorn	NDC 59399-162-35
BCA Protein Assay Kit	Thermo Fisher Scientific	23227	Protein quantification
Biovortexer	Bellco Glass, Inc.	198050000	Hand-held tissue homogenizer
CellSens Software	Olympus	V4.1.1	Image processing to FISH images.
Centrifuge 5810	Eppendorf	EP022628168	Plate centrifuge
Chloroform	Thermo Fisher Scientific	J67241-AP	RNA isolation
Cotton Tipped Applicators	ProAdvantage	77100	Sterilize before use
CRISPR/Cas9 Control Plasmid	Santa Cruz Biotechnology	sc-418922	Dnase-free water provided for dilution
CryoStat	Leica	CM1950
Dissection Microscope	Leica	M125 C	Used for post-necroscopy imaging
Dissolvable Sutures	Med Vet International	J385H
Distilled Water	Gibco	15230162
Dulbecco's Phosphate Buffered Saline (DPBS)	Thermo fisher Scientific	14190144	(-) Calcium; (-) Magnesium
ECM 830 Electro Electroporator (Electroporation Machine)	BTX Harvard Apparatus	45-0662	Generator only
Electric Razor	Wahl	CL9990	Kent Scientific
Electroporation paddles/Tweezertrodes	BTX Harvard Apparatus	45-0487	3 mm diameter paddles; wires included
Embedding Cassette: 250 PK	Grainger	21RK94	Placenta embedding cassettes
Ethanol	Thermo Fisher Scientific	268280010
F-Air Canisters	Penn Veterinary Supply Inc	BIC80120	Excess isoflurane filter
Fast Green Dye FCF	Sigma	F7252-5G	Dissolve to 1 μg/ml and filter; protect from light
Filter-based microplate photometer (plate reader)	Fisher Scientific	14377576	Can be used for BCA and ELISA
Forceps	VWR	82027-386	Fine tips, straight, serrated
Formalin solution, neutral buffered, 10%	Sigma Aldrich	HT501128
Glass Capillaries - Borosilicate Glass (Micropipette)	Sutter Instrument	B150-86-10	O.D.: 1.5 mm, I.D.: 0.86 mm, 10 cm length
Halt Protease and Phosphotase inhibitor cocktail (100x)	Thermo Scientific	1861281	Protein homogenization buffer
Heating Pad	Thermotech	S766D	Digitial Moist Heating Pad
Hemostats	VWR	10806-188	Fully surrated jaw; curved
Hot Water Bath	Fisher Scientific	20253	Isotemp 205
Igf-1 SAM Plasmid (m1)	Santa Cruz Biotechnology	sc-421056-ACT	Dnase-free water provided for dilution
Induction Chamber	Vetamac	941443	No specific liter size required
Isoflurane	Piramal Pharma Limited	NDC 66794-013-25
Isoproponal/2-Proponal	Fisher Scientific	A451-4	RNA isolation
Ketamine HCl 100mg/ml	Akorn	NDC 59399-114-10
MgCl2/Magneisum Chloride	Sigma Aldrich	63069-100ML	1M. Protein homogenization buffer
MicroAmp™ Optical 384-Well Reaction Plate with Barcode	Fisher Scientific	4309849	Barcoded plates not required
Microcapillary Tip	Eppendorf	5196082001	Attached to BTX Microinjector
Microinjector	BTX Harvard Apparatus	45-0766	Stainless Steel Pipette Holder, 130 mm Length, for 1 to 1.5 mm Pipettes
Microject 1000A (Injection Machine)	BTX Harvard Apparatus	45-0751	MicroJect 1000A Plus System
Micropipette Puller Model P-97	Sutter Instrument	P-97	Flaming/Brown type micropipette puller
Microplate Mixer (Plate Shaker)	scilogex	822000049999
Mouse/Rat IGF-I/IGF-1 Quantikine ELISA Kit	R & D Systems	MG100
Needles	BD - Becton, Dickson, and Company	305106	30 Gx 1/2 (0.3 mm x 13 mm)
Nitrogen Tank	Linde	7727-37-9	Any innert gas
Non-Steroidal Anti-Inflammatory Drug (NSAID)	Norbrook Laboratories Limited	NDC 55529-040-10	Analesgic such as Meloxicam
Nose Cone	Vetamac	921609	9-14 mm
Opal 620 detection dye	Akoya Biosciences	SKU FP1495001KT	Used for FISH
Optimal Cutting Temperature (O.C.T) Compound	Sakura	4583
Oxygen Tank	Linde	7782 - 44 - 7	Medical grade oxygen
Pestles	USA Scientific Inc	14155390
Povidone-Iodine Solution, 5%	Avrio Health L.P.	NDC 67618-155-16
Power SYBR™ Green PCR Master Mix	Thermo Fisher Scientific	4367659	Use for qPCR
Random Hexamers (Random Primers)	New England Biolabs	S1330S	Use for cDNA synthesis
Razor Blade	Grainger	26X080
RNA Cleanup Kit & Concentrator	Zymo Research	R1013
RNALater	Thermo Fisher Scientific	AM7021
RNAscope kit v.2.5	Advanced Cells Diagnostics	323100	Contains all reagents required for fluorescent in situ hybridization. Probes sold separately.
RNAscope™ Probe- Mm-Prl8a8-C2	Advanced Cells Diagnostics	528641-C2
RNAscope™ Probe- Vector-dCas9-3xNLS-VP64	Advanced Cells Diagnostics	527421
Roto-Therm Mini	Benchmark	R2020	Dry oven for in situ hybridization
Scissors	VWR	82027-578	Dissecting Scissors, Sharp Tip, 4¹/?
Sodium Chloride (Saline)	Hospra	NDC 0409-4888-03	Sterile,  0.9%
Sodium Citrate, Trisodium Salt, Dihydrate, [Citric Acid, Trisodium Dihydrate]	Research Product International	03-04-6132
Sodium Hydroxide 1N Concentrate, Fisher Chemical	Fisher Scientific	SS277	Protein homogenization buffer
Steamer	Bella	B00DPX8UBA
Sterile Surgical Drape	Busse	696	Sterilize before use
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Surgipath Cover Glass 24x60	Leica	3800160
Syringes	BD - Becton, Dickson, and Company	309659	BD Luer Slip Tip Syringe sterile, single use, 1 mL
Thermo Scientific™ Invitrogen™ Nanodrop™ One Spectrophotometer with WiFi and Qubit™ 4 Fluorometer	Fisher Scientific	13-400-525	This configuration comes with Qubit 4 fluorometer.  Qubit quantification not required.
Tissue Adhesive	3M	1469SB	VetBond
Tris HCl	Thermo Fisher Scientific	15568025	1M. Protein homogenization buffer
TRIzol™ Reagent	Thermo Fisher Scientific	15596018	RNA isolation
TSA Buffer Pack	Advanced Cells Diagnostics	322810	Used to dilute Opal 620 detection dye
Universal F-Circuit	Vetamac	40200	Attached to vaporizer and vaporizer accessories
Upright Compound Fluorescence Microscope	Olympus	BX61VS	Used for FISH imaging
Vectorshield with DAPI	Vector Laboratories	H-1200	Coverslip mounting media
ViiA™ 7 Real-Time PCR System with 384-Well Block	Thermo Fisher Scientific	4453536	This is for SYBR 384-well block detection.  TaqMan and/or smaller blocks available
Wet n Wild Nail Polish Wild Shine, Clear Nail Protector, Nail Color	Amazon	C450B
Xylazine 20mg/ml	Anased	343730_RX