Name: mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis
Uploaded: 2014-09-09T15:00:00
Description: Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Denne undersøgelse blev støttet af NIH R00HL102154, og en American Society of Hematology lærd tildeling til P Ji.

De i denne protokol beskrevne eksperimenter blev udført i overensstemmelse med de retningslinjer og regler, der er fastsat ved Northwestern University Institutional Animal Care og brug Udvalg. 1. Fremstilling af Kultur Medium <ol><li> Forbered fibronektin løsning. Der tilsættes 1 ml vand til et hætteglas af humant fibronectin (1 mg). Lad opløsningen i vævskultur hætte i 30 minutter uden omrøring. Overfør den samlede væske til 50 ml PBS for at gøre en endelig koncentration på 20...

<ol>
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A., Sirin, O., Lin, K. K., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+characterization+and+isolation.">Hematopoietic stem cell characterization and isolation.</a> Methods in molecular biology. 750, 47-59 (2011).</li><li>Choi, H. S., Lee, E. M., Kim, H. O., Park, M. I., Baek, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+control+of+terminal+erythropoiesis+via+physical+interactions+among+erythroid+cells.">Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells.</a> Stem cell research. 10 (3), 442-453 (2013).</li><li>Bhatia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+fatty+acid-mediated+effects+on+erythropoiesis+in+primary+definitive+erythroid+cells.">Short-chain fatty acid-mediated effects on erythropoiesis in primary definitive erythroid cells.</a> Blood. 113 (25), 6440-6448 (2009).</li><li>Zhang, J., Socolovsky, M., Gross, A. W., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ras+signaling+in+erythroid+differentiation+of+mouse+fetal+liver+cells:+functional+analysis+by+a+flow+cytometry-based+novel+culture+system.">Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system.</a> Blood. 102 (12), 3938-3946 (2003).</li><li>Socolovsky, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98 (12), 3261-3273 (2001).</li><li>Ji, P., Jayapal, S. R., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+cultured+mouse+fetal+erythroblasts+requires+Rac+GTPases+and+mDia2.">Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2.</a> Nature cell biology. 10 (3), 314-321 (2008).</li><li>Chida, D., Miura, O., Yoshimura, A., Miyajima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cytokine+signaling+molecules+in+erythroid+differentiation+of+mouse+fetal+liver+hematopoietic+cells:+functional+analysis+of+signaling+molecules+by+retrovirus-mediated+expression.">Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.</a> Blood. 93 (5), 1567-1578 (1999).</li><li>Patel, V. P., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fibronectin+matrix+is+required+for+differentiation+of+murine+erythroleukemia+cells+into+reticulocytes.">A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.</a> The Journal of cell biology. 105 (6 Pt 2), 3105-3118 (1987).</li><li>Udupa, K. B., Lipschitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-induced+suppression+of+erythropoiesis:+the+role+of+erythropoietin+and+a+heme+synthesis+stimulating+factor.">Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor.</a> Blood. 59 (6), 1267-1271 (1982).</li><li>Koury, M. J., Sawyer, S. T., Brandt, S. 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Her præsenterer vi et unikt system til kronologisk analysere mus fosterlever terminal erythropoiese. Gennem anvendelse af forskellige dyrkningsbetingelser, vi med succes dissekeret terminal erytropoiese i tidlige og sene stadier. Dette er især vigtigt for at bestemme de mekanismer af gener med flere funktioner. For eksempel Rac GTPaser spille vigtige roller i forskellige stadier af terminal erythropoiese. Hæmning af Rac GTPaser i den tidlige fase af terminale erytropoiese påvirkninger celledifferentiering og prolife...

Erythropoiese er processen differentiering af multipotente hæmatopoietiske stamceller til modne erythrocytter. Denne trinvise proces omfatter dannelsen af engagerede erythroide bristdannende enheder (BFU-E), de hurtigt delende erythroide kolonidannende enheder (CFU-E), og morfofonologiske genkendelige erythroblaster 1,2. Terminal erytropoiese fra CFU-E stamceller indebærer sekventiel erythropoietin-afhængige og uafhængige trin 2,3. I den tidlige fase af terminal erythropoiese erythropoietin (EPO) bindes til sin receptor på celleoverfladen og fremkalder en række downstream signalveje, der forhindrer celle apoptose og fremme hurtige celledelinger og genekspression 1,4. I den sene fase af terminal erythropoiese erytroblaster undergår terminal cellecyklus exit, kromatin og kerne kondens, og ekstrudering af de stærkt kondenseret kerner 5. Vores forståelse af terminalerythropoiesen har i høj grad forbedret i de seneste årtier, hvilket i høj grad skyldes den vellykkede brug af flere in vitro og in vivo musemodeller 6-9. Blandt disse modeller in vitro-dyrkning af muse føtal lever Erytroblaster giver mange fordele, herunder den lethed celle oprensning, hurtig proliferation og differentiering, og tættere efterligner den menneskelige erythropoiese 10,11. I dette system kan et stort antal af erythroide stamceller fra mus føtale levere let isoleres ved enkelt trin oprensning af TER119 (en markør for de modne erythroide celler) negative erythroblaster. I løbet af to-dages kultur erythroblaster kan differentieringen af disse celler blive overvåget af en flowcytometrisk analyse baseret på overflade ekspression af transferrin-receptoren (CD71) og TER119 antigenet 12. Desuden kan enucleation af terminalt differentiering erytroblaster påvises ved en DNA-maker (Hoechst 33342) 13. Endvidere de oprensede stamceller kan genetisk modificeret ved exogen ekspression af cDNA&#39;er eller små hårnål RNA&#39;er (shRNAs) for generne af interesse, hvilket letter mekanistiske studier af funktionerne af genekspression på erythropoiese 11,13,14. På den anden side kan den hurtige celle vækst være et tveægget sværd, eftersom det er vanskeligt at karakterisere genfunktioner i forskellige stadier af terminal erythropoiese. I de fleste tilfælde er det vanskeligt at afgøre, om et specifikt gen fungerer i den tidlige fase af terminal erythropoiese siden på det tidspunkt, cDNA&#39;erne eller shRNAs udtrykte cellerne allerede passeret den tidlige fase. For at løse dette problem, har vi udviklet et unikt system til at dissekere de tidlige og sene stadier af terminal erythropoiese. For den tidlige fase af terminal erythropoiese genmodificerede TER119 negative erythroblaster blev dyrket i Epo-frit medium, men som indeholder stamceller (SCF), IL-6 og FLT3ligand til at opretholde deres stamfader status og tillade de transducerede cDNA&#39;er eller shRNA ekspression 13. Cellerne blev ændret til Epo medium efter 12 timer for at inducere celleproliferation og differentiering. På denne måde, når cellerne begyndte at skelne de transducerede cDNA&#39;er eller shRNAs allerede udtrykkes. For sent stadium terminal erythropoiese TER119 negative erythroblaster blev dyrket i Epo holdigt medium umiddelbart efter transduktion. Derfor kan man analysere de funktioner af generne af interesse i den sene fase af terminal erythropoiese. Sammenfattende vil en bred anvendelse af dette system hjælpe dissekere genfunktioner i forskellige stadier af terminal erythropoiese.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

Erythropoiesen indebærer en dynamisk proces, der begynder med engagerede erythroid bristdannende enheder (BFU-Es) efterfulgt af hurtigt delende erythroide kolonidannende enheder (CFU-Es). Efter CFU-Es, celler er morfologisk genkendelige og betegnes generelt terminale erythroblaster. En af udfordringerne for studiet af terminalen erythropoiese er manglen på eksperimentelle metoder til at dissekere genfunktioner i kronologisk måde. I denne protokol, beskriver vi en unik strategi for at bestemme genfunktioner i de tidlige og sene stadier af terminal erythropoiese. I dette system blev mus føtal lever TER119 (moden erythroidcelle markør) negative Erytroblaster oprenset og transduceret med eksogene ekspression af cDNA&#39;er eller små hairpin RNA&#39;er (shRNAs) for generne af interesse. Cellerne blev efterfølgende dyrket i medium indeholdende andre end erythropoietin (Epo) for at opretholde deres stamfader fase i 12 timer samtidig med at de eksogene cDNA&#39;er eller shRNAs vækstfaktorerat udtrykke. Cellerne blev ændret til Epo mediet efter 12 timer for at inducere celledifferentiering og proliferation, mens de eksogene genetiske materiale allerede blev udtrykt. Denne protokol letter analyse af gen-funktioner i den tidlige fase af terminal erythropoiese. At studere sent stadium terminal erythropoiese blev cellerne straks dyrket i Epo mediet efter transduktion. På denne måde blev de celler, der allerede differentierede til den sene fase af terminal erythropoiese når de transducerede genetiske materiale blev udtrykt. Vi anbefaler en generel anvendelse af denne strategi, der ville hjælpe med at forstå detaljerede gen funktioner i forskellige stadier af terminal erythropoiese.

Figur 1 skitserer de eksperimentelle strategier. Protokollen består af to uafhængige betingelser for at målrette de funktioner, som signalmolekyler i de tidlige og sene stadier af terminal erythropoiese. TER119 negative fosterleverceller Erytroblaster blev oprenset fra E13.5 mus fosteret. Flowcytometrisk analyse af føtale lever erythroide celler før og efter oprensning viste, at rensningen var effektiv (figur 2A og 2B). På det tidlige stadium af terminale erythrop...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Mus fosterlever Kultur System at dissekere målgenets funktioner på de tidlige og sene stadier af Terminal Erythropoiese

Vi præsenterer et in vitro-mus fosterlever erythroblast kultur, der dissekerer de tidlige og sene stadier af terminal erythropoiese. Dette system letter funktionel analyse af specifikke gener i forskellige udviklingsstadier.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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