Functional Cloning Using a Xenopus Oocyte Expression System

Podsumowanie

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Streszczenie

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Wprowadzenie

Forward genetic approaches to identify genes of interest through their function or loss-of-function^1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers^3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning^6,7.

Our recent aim⁸ was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent⁹ animal cap ectoderm (stage 11-11.5¹⁰) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.

The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland^6,7, also successfully used by others^11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner¹⁴), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm⁸), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb1⁸), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

Protokół

All experimental procedures were approved by the University of Virginia Institutional Animal Care and Use Committee.

Note: Figure 1 shows a schematic overview of the experimental procedures.

1. Preparation of Oocytes

1. Pre-prime X. laevis females with 150 U of Pregnant Mare Serum Gonadotropin (PMSG) approximately one week in advance of oocyte isolation. Inject 1 ml 150 U/ml PMSG into dorsal lymph sac with 1 cc sterile syringe with 29 G needle.
2. Prepare solutions for oocyte injection and oocyte-animal cap assay.
   1. Prepare Ca⁺⁺/Mg⁺⁺-free OR2 (OR2-): 82 mM NaCl, 2.5 mM KCl, 1.5 mM Na₂HPO₄, 50 mM HEPES pH 7.2.
   2. Prepare OR2: OR2- plus 1 mM CaCl₂ and 1 mM MgCl_2.
   3. Prepare OCM: 60% Leibovitz L15, 0.4 mg/ml BSA, 100 μg/ml Gentamycin, pH 7.8.
   4. Prepare 1x MBS: 88 mM NaCl, 1 mM KCl, 0.7 mM CaCl₂, 1 mM MgSO₄, 5 mM HEPES (pH 7.8), 2.5 mM NaHCO_3.
   5. Prepare 3% Ficoll in 1x MBS.
   6. Prepare 1x NAM: 110 mM NaCl, 2 mM KCl, 1 mM Ca(NO₃)₂, 1 mM MgSO₄, 0.1 mM EDTA, 1 mM NaHCO₃, 2 mM Na₃PO₄, pH 7.4.
3. Anesthetize the female in a 0.03% solution of Ethyl 3-aminobenzoate methanesulfonate salt (MS222) in 0.1x MBS (first dissolve 0.3 g MS222 in 10 ml 95% ethanol, then dilute in 1 L 0.1x MBS) by placing frog in solution for 10 - 15 min or until unresponsive.
   1. Check that anesthesia is complete by turning anesthetized frog onto its back to ensure it does not respond (incompletely anesthetized frogs move limbs or turn body over).
4. Surgically isolate ovarian fragments by making an abdominal incision through skin and body wall with scalpel blade, isolating ovarian tissue with forceps and scissors. Place ovarian fragments into OR2-. Close the body wall with a 3-0 silk suture on a 24 mm curved needle and close the skin separately with the same sutures. Allow recovery of female in 1 g/L aquarium salt in water.
5. Using fine forceps, tear ovarian tissue into small (10 - 20 oocytes) pieces and transfer to fresh OR2-.
6. Defolliculate ovarian fragments in 2.0 mg/ml collagenase A in OR2- by agitating gently on shaker for 1 hr, transferring to fresh collagenase and agitating for one additional hour.
7. Wash oocytes 10 times in OR2 [containing Ca⁺⁺/Mg⁺⁺], discarding smaller oocytes.
8. Wash oocytes in OCM two times and transfer to fresh OCM. Isolate Stage VI oocytes by size upon visual inspection and discard immature (smaller) oocytes: Stage VI oocytes are larger than immature oocytes with even pigmentation in the animal hemisphere and are approximately 1.2-1.4 mm in diameter.
9. Maintain oocytes at 18 - 20 °C in OCM prior to injection. Note: Agarose-coated petri dishes may be used to minimize sticking of oocytes to dish.

2. Injection of Library Transcripts

1. Prepare a directional cDNA library¹⁵ using RNA extracted at an appropriate stage of development (for example, neural plate stage 14¹⁰). Purchase a cDNA library or construct one using a commercial kit per the overview in the four steps below.
   1. Isolate approximately 5 μg mRNA by using a product to enrich for poly(A)+ RNA (See Table of Materials) and following manufacturer's instructions. Note: The transcripts to be used to produce the cDNA library may be restricted to an inducing tissue (such as the neural plate, following microdissection of the desired tissue), or may be from whole embryos at a particular stage.
   2. Produce cDNA with a commercial kit, following manufacturer's directions.
   3. Ligate approximately 20 ng cDNA into a vector included with the commercial kit or another suitable vector. An appropriate plasmid vector includes sequences for mRNA stability such as 5' β-globin and 3 poly(A) sequences (pCS2+, pTnT, pCS105).
   4. Transform ligation into competent bacterial cells using supplier's recommended protocol for heat shock transformation.
      Note: Alternatively, a collection of open reading frame (ORFeome) clones is available commercially and may be used to generate transcripts for injection.
2. Prepare 10 pools of plasmids of 10³ to 10⁴ complexity (10⁴ to 10⁵ total complexity). This represents 10 plates with 1,000 - 10,000 colonies each.
   1. Plate library culture onto 10 15-cm LB-ampicillin plates, grow 12 - 18 hr at 37 ˚C, and collect colonies from each by gentle pressure with a glass spreader in 7 ml LB.
   2. Prepare a glycerol stock from 0.5 ml (add to 0.2 ml sterile glycerol and store at -20 ˚C), and use the remaining 6.5 ml to prepare DNA with a standard commercially available DNA miniprep kit following manufacturer's directions.
3. Linearize pooled plasmid DNA (1.0 - 2.0 μg) with appropriate restriction enzyme digest¹⁶ at 37 ˚C for 1 - 1.5 hr. Isolate the linearized DNA with phenol/chloroform extraction followed by ethanol precipitation and resuspension in water per the specifications of the RNA polymerase kit used. Synthesize sense RNA with a commercial RNA polymerase kit following manufacturer's directions.
4. Prepare needles for microinjection (using a needle puller with glass capillary tubing) of approximately 20 μm diameter; measure needle tips on a compound microscope with a calibrated ocular micrometer. Note: Needle puller settings must be empirically determined to produce a needle with a fine tip such that when broken with fine forceps yields the desired tip size.
5. Prepare (push clay into uniform layer on bottom of dish) clay-lined 35 x 10 mm petri dishes with parallel grooves to hold oocytes in place during microinjection; produce grooves with a mall probe or Pasteur pipette fused at the tip in flame. As an alternative to clay, prepare agarose dishes making indentations with an elastomer mold¹⁷. Transfer oocytes with a wide-bore pipette to 3% Ficoll in 1X MBS (approximately 2 ml) in rows in the clay-lined dishes.
6. Using microinjector, fill needle with 1 ng/nl RNA and adjust balance to produce slight positive pressure (to prevent drawing up of oocyte cytoplasm).
7. Inject oocytes with approximately 20 nl RNA in the equatorial region. Allow 1 hr for injected oocytes to remain in Ficoll-MBS, then transfer gently to 1x MBS. Incubate for 8 - 24 hr at 20 °C prior to animal cap assay.

3. Animal Cap Assay

1. Prepare 3/4x NAM and obtain fine forceps, hair loop, curved coverslip fragments, clay-lined dishes with cup-shaped indentations to hold oocytes. Make curved coverslip fragments by breaking apart glass coverslips into small fragments (approximately 1 - 2 mm x 2 - 4 mm) and passing through flame until edges polish and droop, producing a curved piece.
2. Fertilize X. laevis eggs¹⁸ through in vitro or natural mating and culture to gastrula stages (10 - 11 hr post-fertilization at RT) in 0.1x MBS prior to sorting by stage; collect mid-gastrula (stage 11 - 11.5)¹⁰ embryos.
3. Transfer embryos to a petri dish approximately half-full with 3/4x NAM. Using two pairs of fine forceps, remove fertilization (vitelline) membrane from gastrulae.
4. Transfer oocytes to 3/4x NAM (approximately 2 ml) in clay-lined dishes and immobilize in individual impressions in the clay, producing impressions to accommodate individual embryos as grooves were described above.
5. Transfer embryos to the clay-lined dishes and cut animal caps off gastrulae using two pairs of fine forceps. Take care to isolate animal cap ectoderm only and not equatorial tissue¹⁸.
6. Place an animal cap on the animal hemisphere of each oocyte with the inner surface of the animal cap contacting the oocyte. Hold recombinants together by applying curved glass coverslip fragment and applying downward pressure to the glass, flattening the ectoderm as the coverslip contacts the clay (animal caps can remain open with the inner layer exposed to the surface of the oocyte for 6 - 8 hr or longer).
   Note: Alternatively, place the animal cap into a clay indentation with its inner surface facing upward and place an oocyte on the cap; secure the recombinant with small extensions of clay.
7. Culture at 20 °C until control embryos reach desired stage for assay.
8. Separate recombinant by removing coverslip/clay and isolating ectoderm with forceps and a hair loop.
9. Fix ectodermal fragments for 1 hr in MEMFA (3.8% formaldehyde in MEM [0.1 M MOPS pH 7.4, 2 mM EGTA, and 1 mM MgSO₄]).
10. Transfer fragments (and control embryos) from MEMFA into ethanol and store at -20 ˚C.

4. Analysis of Response in Ectoderm by In situ Hybridization (ISH)

1. Prepare solutions for ISH.
   1. Prepare 1x PBS: 0.01 M phosphate buffered saline, NaCl 0.138 M, pH 7.4
   2. Prepare PBS-Tween (PTw): 1x PBS, 0.1% Tween-20
   3. Prepare 100x Denhart's solution: 2% BSA, 2% Polyvinylpyrrolidone, 2% Ficoll
   4. Prepare Hybridization Buffer: 50% Formamide, 5x SSC, 1 mg/ml torula yeast RNA, 1 μg/mL Heparin, 1x Denhart's solution, 0.1% Tween-20, 0.1% CHAPS, 10 mM EDTA, DEPC-H₂O
   5. Prepare Maleic Acid Buffer (MAB): 100 mM maleic acid, 150 mM NaCl, pH 7.5
   6. Prepare MAB + block: MAB, 2% blocking reagent (heat to 60 ˚C to dissolve)
   7. Prepare Alkaline Phosphatase (AP) Buffer: 100 mM Tris pH 9.5, 50 mM MgCl₂, 100 mM NaCl, 0.1% Tween, dH₂O
2. Prepare the RNA Probe.
   1. Using a commercial RNA polymerase kit and dig-NTP mix, add to a 1.5 ml tube (50 μl reaction): 25.5 μl DEPC-H₂O, 10 μl 5X Transcription Buffer, 2.5 μl 10x dig-NTP mix, 5 μl 100 mM DTT, 2 μl RNAsin, 2 μl linearized DNA template (~1 μg/μl), 3 μl RNA Polymerase and incubate 37 ˚C for 90 min.
   2. Add 2 μl RNA Polymerase and incubate 37 ˚C for 60 min.
   3. Check 2 μl of reaction on 1% agarose gel.
   4. Add 1 μl RQ1 RNase-Free DNase and incubate 37 ˚C for 20 min.
   5. Precipitate the probe by adding 50 μl DEPC-H₂O, 25 μl 10 M Ammonium Acetate and 313 μl ethanol. Store at -20 ˚C overnight (O/N) then recover RNA by centrifugation at 13,800 x g for 20 min. Wash with 500 μl 75% ethanol, spin briefly, remove ethanol and allow pellet to air dry. Add 50 μl DEPC-H₂O.
   6. Add Hybridization Buffer to a final concentration of ~0.5 μg/μl.
3. Prepare Tissue for Hybridization. Unless otherwise noted, fill vials to the top with each solution change described (approximately 4 ml).
   1. Remove ethanol from vials and transfer embryos into 75% ethanol/PTw, then 50% ethanol/PTw for 10 min each, horizontally on rocker.
   2. Wash three times in PTw for 5 min each on rocker.
   3. Transfer to 10 μg/μl Proteinase K treatment in PTw; rock tubes vertically 15 min.
   4. Rinse twice 10 min each in 0.1 M Triethanolamine pH 7.8 - rock tubes vertically.
   5. Add 12.5 μl acetic anhydride to tubes and rock vertically 5 min. Repeat with additional 12.5 μl acetic anhydride for 5 min.
   6. Wash in PTw 5 min vertically on rocker.
   7. Refix in 4% paraformaldehyde 20 min on rocker. Heat Hybridization Buffer to 60 ˚C.
   8. Wash three times in PTw for 5 min each on rocker.
   9. Remove all but ~1 ml of PTw from each tube and add 250 μl Hyb Buffer; gently swirl tubes to mix. Rock tubes vertically 5 min.
   10. Replace with 60 ˚C Hyb Buffer (0.5 ml) and agitate gently at 60 ˚C 10 min. Replace with fresh Hyb Buffer and agitate at 60 ˚C two to 4 hr.
   11. Heat probe (1 ml at 0.5 μg/μl in Hyb Buffer) to 60 ˚C (3 min). Remove Hyb Buffer and add probe to tubes. Agitate gently O/N at 60 ˚C.
4. Prepare Tissue for Antibody.
   1. Warm Hyb Buffer and 2x SSC + 0.1% Tween-20 solutions to 60 ˚C.
   2. Replace probe solution with Hyb Buffer (save probes at -20 ˚C for 2 - 3x reuse). Wash at 60 ˚C for 10 min.
   3. Wash three times at 60 ˚C in 2x SSC-Tween (20 min each with agitation).
   4. Wash three times at 60 ˚C in 0.2x SSC-Tween (20 min each with agitation).
   5. Wash two times in MAB (RT) for 15 min each horizontally on rocker.
   6. Add 1 ml MAB + block. Wash 2 hr vertically on rocker.
   7. Transfer to 1 ml MAB + block containing a 1/2,000 Anti-digoxygenin-AP. Rock vertically at 4 ˚C O/N.
5. Prepare Tissue for Color Reagent.
   1. Replace antibody solution with MAB; wash three times in MAB 5 min each horizontally on rocker.
   2. Wash three times in MAB 1 hr each horizontally on rocker.
   3. Wash two times in AP Buffer 10 min each horizontally on rocker.
   4. Transfer tissue to multiple-well plastic tray, remove AP Buffer and add BM Purple reagent (~1 ml). Allow reaction to proceed in dark 5 min to O/N.
   5. When appropriate staining is achieved, transfer tissue to vials of PTw. Wash two times 10 min each in PTw on rocker.
   6. Fix in Bouin's solution at 4 ˚C O/N on rocker.
   7. Wash out Bouin's with three 70% ethanol/30% PTw washes at RT. Proceed to image capture using epi-illumination and a microscope-mounted camera, bleaching if necessary¹⁸.

5. Sib Selection and Cloning

1. Select pool with the highest activity (greatest response in ectodermal tissue).
2. Titrate (dilute with LB to appropriate density) the glycerol stock for the pool with the highest activity and plate out ten new plates with approximately one-tenth the colonies from the previous step (using section 2 in protocol above).
3. Reduce pool size until activity is traced to single colony.
4. Obtain DNA sequence using standard primers in the vector.

Wyniki

In response to expression of mRNA injected into oocytes, responding animal cap tissue was assayed for expression of otx2 by in situ hybridization (Figure 2 and Table 1); otx2 is expressed in the presumptive lens ectoderm (PLE) from neural tube closure through lens placode thickening¹⁹. However, since otx2 is also expressed in the anterior neural ectoderm as well as non-neural head ectoderm outside the PLE, it is associated wi...

Dyskusje

The method described here for the functional cloning of genes capable of inducing a response in competent ectoderm can be used to identify a wide range of gene products. This method expands upon past work by combining tissue-inducing assays with expression cloning techniques. We utilize the metabolic pathways of the Xenopus oocyte as a source of production of inducing factors, directly or indirectly, following RNA injection. This, in combination with the use of established methods for cloning a gene of interest<...

Materiały

Name	Company	Catalog Number	Comments
12/101 Antibody	Developmental Studies Hybridoma Bank	12/101	Monoclonal antibody for detection of muscle tissue
20x SSC Buffer	Sigma	S6639	for ISH
Acetic anhydride	Sigma	A6404	for ISH
Anti-Dig-AP	Roche	11093274910	for ISH
Aurum Plasmid Mini Kit	Bio-Rad	732-6400	Plasmid DNA purification
Blocking Reagent	Roche	11096176001	for ISH
BM Purple	Roche	11442074001	for ISH
Boekel Hybridization Oven	Fisher Scientific	13-245-121	for ISH
Bouin's Solution	Sigma	HT10132	for ISH
BSA	Sigma	A9647	for OCM
CHAPS	Sigma	C3023	for ISH
Collagenase A	Roche	10103578001	Defolliculation of oocytes
Cysteine	Sigma	C121800	Dejelly embryos
DEPC-H2O	Fisher Scientific	BP5611	for ISH
Dig-RNA Labeling Mix	Roche	11277073910	for ISH probes
Dumont #5 forceps	World Precision Instruments	500233	for Vitelline envelope removal
Ethyl 3-aminobenzoate	Sigma	A5040	MS222 anesthetic
Ficoll PM 400	Sigma	F4375	for Injection media
Formamide	Sigma	F9037	for ISH
Gentamicin sulfate	Sigma	G1914	for OCM
Glass capillaries	World Precision Instruments	4878	3.5" long, I,D, 0.530 mm
Glass sample vials	Fisher Scientific	06-408B	for ISH
Hair loop			Hair affixed in pasteur pipette for tissue manipulation
Heparin sodium salt	Sigma	H4784	for ISH
Injector Nanoliter 2010	World Precision Instruments	Nanoliter 2010	Microprocessor-controlled microinjector
Instant Ocean	Carolina	972433	Aquarium Salt for frog recovery
IRBG XGC Xenopus verified full-length cam cDNA	Source Bioscience	989_IRBG	cDNA library
LB Agar plates with 100 µg/ml Ampicillin	Teknova	L5004	150 mm pre-poured LB-Amp plates for sib selection
LB Luria Broth	Teknova	L8650	LB for collecting colonies in sib selection from plates and dilution of cultures
Magnetic mRNA Isolation Kit	New England BioLabs	S1550S	for isolation of poly(A)-enriched RNA
Maleic Acid	Sigma	M0375	for ISH
Manual Microfil Micromanipulator	World Precision Instruments	M3310R	Manual micromanipulator
Nutating Mixer	Fisher Scientific	22-363-152	Rocker for ISH
Permoplast	Nasco	SB33495M	Clay for injection and dissection dishes
Phosphate Buffered Saline	Sigma	P5368	for ISH
PMSG	Sigma	G4877	to stimulate oocyte development
Polyvinylpyrrolidone	Sigma	PVP40	for ISH
Programmable Puller	World Precision Instruments	PUL-1000	Micropipette needle puller
Proteinase K	Sigma	P6556	for ISH
pTnT Vector	Promega	L5610	cDNA library construction
Riboprobe Combination System	Promega	P1450	in vitro transcription
Superscript Full Length cDNA Library Construction Kit	Life Technologies	18248013	kit for cDNA library construction
Sutures, 3-0 silk	Fisher Scientific	19-037-516	Suture thread and needle for post-oocyte removal
Torula RNA	Sigma	R3629	for ISH
Triethanolamine	Sigma	T1502	for ISH
Tween 20	Sigma	P9416	for ISH
Universal RiboClone cDNA Synthesis System	Promega	C4360	alternative kit for cDNA library construction
Xenopus Full ORF Entry Clones - ORFeome Collaboration	Source Bioscience	5055_XenORFeome	ORFeome Clones
XL2-Blue Ultracompetent Cells	Agilent Technologies	200150	cells for transformation of cDNA library