This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

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
<ol>
	<li>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.</li>
	<li>Prepare solutions for oocyte injection and oocyte-animal cap assay.
	<ol>
		<li>Prepare Ca++/Mg++-free OR2 (OR2-): 82 mM NaCl, 2.5 mM KCl, 1.5 mM Na2HPO4, 50 mM HEPES pH 7.2.</li>
		<li>Prepare OR2: OR2- plus 1 mM CaCl2 and 1 mM MgCl2.</li>
		<li>Prepare OCM: 60% Leibovitz L15, 0.4 mg/ml BSA, 100 &#956;g/ml Gentamycin, pH 7.8.</li>
		<li>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 NaHCO3.</li>
		<li>Prepare 3% Ficoll in 1x MBS.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Using fine forceps, tear ovarian tissue into small (10 - 20 oocytes) pieces and transfer to fresh OR2-.</li>
	<li>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.</li>
	<li>Wash oocytes 10 times in OR2 [containing Ca++/Mg++], discarding smaller oocytes.</li>
	<li>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.</li>
	<li>Maintain oocytes at 18 - 20 &#176;C in OCM prior to injection. Note: Agarose-coated petri dishes may be used to minimize sticking of oocytes to dish.</li>
</ol>
2. Injection of Library Transcripts
<ol>
	<li>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.
	<ol>
		<li>Isolate approximately 5 &#956;g mRNA by using a product to enrich for poly(A)+ RNA (See Table of Materials) and following manufacturer&#39;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.</li>
		<li>Produce cDNA with a commercial kit, following manufacturer&#39;s directions.</li>
		<li>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&#39; &#946;-globin and 3 poly(A) sequences (pCS2+, pTnT, pCS105).</li>
		<li>Transform ligation into competent bacterial cells using supplier&#39;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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Plate library culture onto 10 15-cm LB-ampicillin plates, grow 12 - 18 hr at 37 &#730;C, and collect colonies from each by gentle pressure with a glass spreader in 7 ml LB.</li>
		<li>Prepare a glycerol stock from 0.5 ml (add to 0.2 ml sterile glycerol and store at -20 &#730;C), and use the remaining 6.5 ml to prepare DNA with a standard commercially available DNA miniprep kit following manufacturer&#39;s directions.</li>
	</ol>
	</li>
	<li>Linearize pooled plasmid DNA (1.0 - 2.0 &#956;g) with appropriate restriction enzyme digest16 at 37 &#730;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&#39;s directions.</li>
	<li>Prepare needles for microinjection (using a needle puller with glass capillary tubing) of approximately 20 &#956;m 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.</li>
	<li>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.</li>
	<li>Using microinjector, fill needle with 1 ng/nl RNA and adjust balance to produce slight positive pressure (to prevent drawing up of oocyte cytoplasm).</li>
	<li>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 &#176;C prior to animal cap assay.</li>
</ol>
3. Animal Cap Assay
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Culture at 20 &#176;C until control embryos reach desired stage for assay.</li>
	<li>Separate recombinant by removing coverslip/clay and isolating ectoderm with forceps and a hair loop.</li>
	<li>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]).</li>
	<li>Transfer fragments (and control embryos) from MEMFA into ethanol and store at -20 &#730;C.</li>
</ol>
4. Analysis of Response in Ectoderm by In situ Hybridization (ISH)
<ol>
	<li>Prepare solutions for ISH.
	<ol>
		<li>Prepare 1x PBS: 0.01 M phosphate buffered saline, NaCl 0.138 M, pH 7.4</li>
		<li>Prepare PBS-Tween (PTw): 1x PBS, 0.1% Tween-20</li>
		<li>Prepare 100x Denhart&#39;s solution: 2% BSA, 2% Polyvinylpyrrolidone, 2% Ficoll</li>
		<li>Prepare Hybridization Buffer: 50% Formamide, 5x SSC, 1 mg/ml torula yeast RNA, 1 &#956;g/mL Heparin, 1x Denhart&#39;s solution, 0.1% Tween-20, 0.1% CHAPS, 10 mM EDTA, DEPC-H2O</li>
		<li>Prepare Maleic Acid Buffer (MAB): 100 mM maleic acid, 150 mM NaCl, pH 7.5</li>
		<li>Prepare MAB + block: MAB, 2% blocking reagent (heat to 60 &#730;C to dissolve)</li>
		<li>Prepare Alkaline Phosphatase (AP) Buffer: 100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween, dH2O</li>
	</ol>
	</li>
	<li>Prepare the RNA Probe.
	<ol>
		<li>Using a commercial RNA polymerase kit and dig-NTP mix, add to a 1.5 ml&#160;tube (50 &#956;l reaction): 25.5 &#956;l DEPC-H2O, 10 &#956;l 5X Transcription Buffer, 2.5 &#956;l 10x dig-NTP mix, 5 &#956;l 100 mM DTT, 2 &#956;l RNAsin, 2 &#956;l linearized DNA template (~1 &#956;g/&#956;l), 3 &#956;l RNA Polymerase and incubate 37 &#730;C for 90 min.</li>
		<li>Add 2 &#956;l RNA Polymerase and incubate 37 &#730;C for 60 min.</li>
		<li>Check 2 &#956;l of reaction on 1% agarose gel.</li>
		<li>Add 1 &#956;l RQ1 RNase-Free DNase and incubate 37 &#730;C for 20 min.</li>
		<li>Precipitate the probe by adding 50 &#956;l DEPC-H2O, 25 &#956;l 10 M Ammonium Acetate and 313 &#956;l ethanol. Store at -20 &#730;C overnight (O/N)&#160;then recover RNA by centrifugation at 13,800 x g for 20 min. Wash with 500 &#956;l 75% ethanol, spin briefly, remove ethanol and allow pellet to air dry. Add 50 &#956;l DEPC-H2O.</li>
		<li>Add Hybridization Buffer to a final concentration of ~0.5 &#956;g/&#956;l.</li>
	</ol>
	</li>
	<li>Prepare Tissue for Hybridization. Unless otherwise noted, fill vials to the top with each solution change described (approximately 4 ml).
	<ol>
		<li>Remove ethanol from vials and transfer embryos into 75% ethanol/PTw, then 50% ethanol/PTw for 10 min each, horizontally on rocker.</li>
		<li>Wash three times in PTw for 5 min each on rocker.</li>
		<li>Transfer to 10 &#956;g/&#956;l Proteinase K treatment in PTw; rock tubes vertically 15 min.</li>
		<li>Rinse twice 10 min each in 0.1 M Triethanolamine pH 7.8 - rock tubes vertically.</li>
		<li>Add 12.5 &#956;l acetic anhydride to tubes and rock vertically 5 min. Repeat with additional 12.5 &#956;l acetic anhydride for 5 min.</li>
		<li>Wash in PTw 5 min vertically on rocker.</li>
		<li>Refix in 4% paraformaldehyde 20 min on rocker. Heat Hybridization Buffer to 60 &#730;C.</li>
		<li>Wash three times in PTw for 5 min each on rocker.</li>
		<li>Remove all but ~1 ml of PTw from each tube and add 250 &#956;l Hyb Buffer; gently swirl tubes to mix. Rock tubes vertically 5 min.</li>
		<li>Replace with 60 &#730;C Hyb Buffer (0.5 ml) and agitate gently at 60 &#730;C 10 min. Replace with fresh Hyb Buffer and agitate at 60 &#730;C two to 4 hr.</li>
		<li>Heat probe (1 ml at 0.5 &#956;g/&#956;l in Hyb Buffer) to 60 &#730;C (3 min). Remove Hyb Buffer and add probe to tubes. Agitate gently O/N at 60 &#730;C.</li>
	</ol>
	</li>
	<li>Prepare Tissue for Antibody.
	<ol>
		<li>Warm Hyb Buffer and 2x SSC + 0.1% Tween-20 solutions to 60 &#730;C.</li>
		<li>Replace probe solution with Hyb Buffer (save probes at -20 &#730;C for 2 - 3x&#160;reuse). Wash at 60 &#730;C for 10 min.</li>
		<li>Wash three times at 60 &#730;C in 2x SSC-Tween (20 min each with agitation).</li>
		<li>Wash three times at 60 &#730;C in 0.2x SSC-Tween (20 min each with agitation).</li>
		<li>Wash two times in MAB (RT) for 15 min each horizontally on rocker.</li>
		<li>Add 1 ml MAB + block. Wash 2 hr vertically on rocker.</li>
		<li>Transfer to 1 ml MAB + block containing a 1/2,000 Anti-digoxygenin-AP. Rock vertically at 4 &#730;C O/N.</li>
	</ol>
	</li>
	<li>Prepare Tissue for Color Reagent.
	<ol>
		<li>Replace antibody solution with MAB; wash three times in MAB 5 min each horizontally on rocker.</li>
		<li>Wash three times in MAB 1 hr each horizontally on rocker.</li>
		<li>Wash two times in AP Buffer 10 min each horizontally on rocker.</li>
		<li>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.</li>
		<li>When appropriate staining is achieved, transfer tissue to vials of PTw. Wash two times 10 min each in PTw on rocker.</li>
		<li>Fix in Bouin&#39;s solution at 4 &#730;C O/N on rocker.</li>
		<li>Wash out Bouin&#39;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.</li>
	</ol>
	</li>
</ol>
5. Sib Selection and Cloning
<ol>
	<li>Select pool with the highest activity (greatest response in ectodermal tissue).</li>
	<li>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).</li>
	<li>Reduce pool size until activity is traced to single colony.</li>
	<li>Obtain DNA sequence using standard primers in the vector.</li>
</ol>

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The authors declare that they have no competing financial interests.

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<...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 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 researchers3-5, 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 cloning6,7.
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 Harland6,7, also successfully used by others11-13. 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.

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">12/101 Antibody</td>
			<td style="width:191px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:135px;">12/101</td>
			<td style="width:233px;">Monoclonal antibody for detection of muscle tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">20X SSC Buffer</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">S6639</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Acetic anhydride</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A6404</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Anti-Dig-AP</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11093274910</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Aurum Plasmid Mini Kit</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:135px;">732-6400</td>
			<td style="width:233px;">Plasmid DNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Blocking Reagent</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11096176001</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">BM Purple</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11442074001</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Boekel Hybridization Oven</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">13-245-121</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Bouin&#39;s Solution</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">HT10132</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">BSA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A9647</td>
			<td style="width:233px;">for OCM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">CHAPS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C3023</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Collagenase A</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">10103578001</td>
			<td style="width:233px;">Defolliculation of oocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cysteine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C121800</td>
			<td style="width:233px;">Dejelly embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">DEPC-H2O</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">BP5611</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Dig-RNA Labeling Mix</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11277073910</td>
			<td style="width:233px;">for ISH probes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Dumont #5 forceps</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">500233</td>
			<td style="width:233px;">for Vitelline envelope removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ethyl 3-aminobenzoate</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A5040</td>
			<td style="width:233px;">MS222 anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ficoll PM 400</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F4375</td>
			<td style="width:233px;">for Injection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Formamide</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F9037</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Gentamicin sulfate</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G1914</td>
			<td style="width:233px;">for OCM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Glass capillaries</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">4878</td>
			<td style="width:233px;">3.5&#34; long, I,D, 0.530mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glass sample vials</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">06-408B</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Hair loop</td>
			<td style="width:191px;"></td>
			<td style="width:135px;"></td>
			<td style="width:233px;">Hair affixed in pasteur pipette for tissue manipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Heparin sodium salt</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">H4784</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Injector Nanoliter 2010</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">Nanoliter 2010</td>
			<td style="width:233px;">Microprocessor-controlled microinjector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Instant Ocean</td>
			<td style="width:191px;">Carolina</td>
			<td style="width:135px;">972433</td>
			<td style="width:233px;">Aquarium Salt for frog recovery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">IRBG XGC Xenopus verified full-length cam cDNA</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">989_IRBG</td>
			<td style="width:233px;">cDNA library&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">LB Agar plates with 100 &#181;g/mL Ampicillin</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L5004</td>
			<td style="width:233px;">150mm pre-poured LB-Amp plates for sib selection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">LB Luria Broth</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L8650</td>
			<td style="width:233px;">LB for collecting colonies in sib selection from plates and dilution of cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Magnetic mRNA Isolation Kit</td>
			<td style="width:191px;">New England BioLabs</td>
			<td style="width:135px;">S1550S</td>
			<td style="width:233px;">for isolation of poly(A)-enriched RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Maleic Acid</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">M0375</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Manual Microfil Micromanipulator</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">M3310R</td>
			<td style="width:233px;">Manual micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Nutating Mixer</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">22-363-152</td>
			<td style="width:233px;">Rocker for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Permoplast</td>
			<td style="width:191px;">Nasco</td>
			<td style="width:135px;">SB33495M</td>
			<td style="width:233px;">Clay for injection and dissection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate Buffered Saline</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P5368</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PMSG</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G4877</td>
			<td style="width:233px;">to stimulate oocyte development</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Polyvinylpyrrolidone</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">PVP40</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Programmable Puller</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">PUL-1000</td>
			<td style="width:233px;">Micropipette needle puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Proteinase K</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P6556</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">pTnT Vector</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">L5610</td>
			<td style="width:233px;">cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Riboprobe Combination System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">P1450</td>
			<td style="width:233px;">in vitro transcription</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Superscript Full Length cDNA Library Construction Kit</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:135px;">18248013</td>
			<td style="width:233px;">kit for cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Sutures, 3-0 silk</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">19-037-516</td>
			<td style="width:233px;">Suture thread and needle for post-oocyte removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Torula RNA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">R3629</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Triethanolamine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">T1502</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Tween 20</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P9416</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Universal RiboClone cDNA Synthesis System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">C4360</td>
			<td style="width:233px;">alternative kit for cDNA library construction</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">Xenopus Full ORF Entry Clones - ORFeome Collaboration</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">5055_XenORFeome</td>
			<td style="width:233px;">ORFeome Clones</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">XL2-Blue Ultracompetent Cells</td>
			<td style="width:191px;">Agilent Technologies</td>
			<td style="width:135px;">200150</td>
			<td style="width:233px;">cells for transformation of cDNA library</td>
		</tr>
	</tbody>
</table>

functional cloning using a xenopus oocyte expression system

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.

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...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

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.

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

functional-cloning-using-a-xenopus-oocyte-expression-system

Research

JoVE Journal

Developmental Biology

8.0K Views.  Shepherd University. 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.

Video: Functional Cloning Using a Xenopus Oocyte Expression System

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...