The authors acknowledge the Foundation Leducq (MITRAL) and the Agence Nationale pour la Recherche (grant ANR Specistem) for funding this research.

1. Preparation
Animal procedures
Obtain approval from an animal ethical committee and follow institutional guidelines for work with virus, HuESC and/or iPSC (when applicable), as well as mouse handling, obtaining mouse embryos, and performing mouse surgery. For timed matings, the day of the plug is considered embryonic day (E)0.5 / 0.5 days post-coitum.
<ol>
	<li>Microinjection needles: 
	For ex utero microinjection, pull glass pipettes (1.2 mm exter...

<ol>
	<li>Thomson, J. A., 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Mummery, C. L., 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=Differentiation+of+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells+to+cardiomyocytes:+a+methods+overview.">Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.</a> Circ. Res. 111, 344-358 (2012).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annu. Rev. Biomed. Eng. 13, 27-53 (2011).</li><li>Dickinson, L. E., Kusuma, S., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructing+the+differentiation+niche+of+embryonic+stem+cells+using+biomaterials.">Reconstructing the differentiation niche of embryonic stem cells using biomaterials.</a> Macromol. Biosci. 11, 36-49 (2011).</li><li>Van Vliet, P., Zaffran, S., Wu, S., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+cardiac+development:+a+view+from+stem+cells+to+embryos.">Early cardiac development: a view from stem cells to embryos.</a> Cardiovasc. Res. In press, (2012).</li><li>Cai, C. L., 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=A+myocardial+lineage+derives+from+Tbx18+epicardial+cells.">A myocardial lineage derives from Tbx18 epicardial cells.</a> Nature. 454, 104-108 (2008).</li><li>Boulland, J. L., Halasi, G., Kasumacic, N., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+human+stem+cells+into+the+chicken.">Xenotransplantation of human stem cells into the chicken.</a> J. Vis. Exp. , (2010).</li><li>Liu, A., Joyner, A. L., Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+limb+and+brain+patterning+in+early+mouse+embryos+by+ultrasound-guided+injection+of+Shh-expressing+cells.">Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells.</a> Mech. Dev. 75, 107-115 (1998).</li><li>Pierfelice, T. J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+microinjection+into+the+mouse+forebrain+in+utero+at+E9.5.">Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.</a> J. Vis. Exp. , (2010).</li><li>Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase:+the+universal+reagent+for+genome+tailoring.">Cre recombinase: the universal reagent for genome tailoring.</a> Genesis. 26, 99-109 (2000).</li><li>Buckingham, M. E., Meilhac, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+cells+for+tracking+cell+lineage+and+clonal.">Tracing cells for tracking cell lineage and clonal.</a> Dev. Cell. 21, 394-409 (2011).</li><li>Phoon, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tools+for+the+developmental+biologist:+ultrasound+biomicroscopy+of+mouse+embryonic+development.">Imaging tools for the developmental biologist: ultrasound biomicroscopy of mouse embryonic development.</a> Pediatr. Res. 60, 14-21 (2006).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat. Protoc. 1, 241-245 (2006).</li><li>Dyer, L. A., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Ex+vivo+Culture+Method+for+the+Embryonic+Mouse.">A Novel Ex vivo Culture Method for the Embryonic Mouse.</a> J. Vix. Exp. , (2013).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+a+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.</a> Dev. Biol. 95, 108-114 (1983).</li></ol>

The authors have nothing to disclose.

The intra-cardiac ex vivo injection protocols described above are designed to preserve myocardial function for at least 48 hr in mid-stage (E9.5- E11.5) mouse embryos. These injection approaches allow for spatially targeted injection of DNA or cells. The few examples shown in Figures 1-3 provide proof of concept for delineating ex vivo and in vivo molecular mechanisms of developmental processes that take place in restricted cardiac regions, such as EMT of endocardial or epicard...

More than a decade ago, human embryonic stem cells (HuESCs) have been derived from human blastocysts1. Since then, these cells have become the subject of an important field of research which addresses unmet questions in human developmental biology. HuESCs have furthermore provided hopes in regenerative medicine. In recent years, human induced pluripotent stem cells (iPSCs) have been generated from patient-specific somatic cells, providing models of genetic disease2. Many in vitro protocols for differentiation of embryonic or induced pluripotent stem cells towards various cell lineages, including heart lineages3, have been reported. The differentiated cells are often phenotyped by analysis of RNA and protein expression, immunostaining, and/or in vitro functional tests. However, pluripotent stem cell derivatives have to be placed in a proper embryonic environment in order to test whether they fully acquire the cell fate of their embryonic counterpart and whether they recapitulate the genuine in vivo function in response to regional cues. While tissue engineering is promising, it does not yet provide all the known and unknown cues of the proper in vivo developing embryonic tissue4,5.
Cell labeling with dyes or retroviral vectors in embryos, including mouse embryos, have brought important information as to the embryonic origin of cell lineages during cardiac development6. For example, dye injection into the pericardial space of mouse embryos ex vivo, followed by in vitro culture of isolated hearts, was used to label epicardial cells and their descendants7. However, dye and retroviral cell labeling have been mostly applied to early mouse embryos with still poorly developed organs, or chicken embryos, which are more easily accessible8. An exception is the brain, which is easier to target in embryos9,10. Such an approach has not yet been applied to the beating embryonic mouse heart.
To complement direct labeling with dyes or virus and to perform lineage tracing in more advanced stage mouse embryos and adult mice, the cell labeling approach has been combined with analysis of transgenic mice using the Cre/Lox technology. The Cre/Lox approach11 however features some limitations due to the spatiotemporal specificity of the genomic regulatory regions used to drive expression of the recombinase, and the efficiency of the Cre/Lox recombination12. Furthermore, this approach does not fully address the specific questions of cell migration-driven acquisition of cell fate as it can only label a precursor after activation of the regulatory region used to drive Cre expression. It also&#160;cannot apply to human embryos for obvious ethical issues.
Given these limitations, we designed a series of new protocols to inject a variety of cell labeling agents such as fluorescent dyes, virus, gene expression modulators such as shRNAs and DNA-based cell labeling vectors, or cells in the mouse embryo at E9.5 and later stages of development in targeted regions of the heart.
The DNA/cell injections use a stereomicroscope and a simple microinjection device combined with ex vivo embryo culture up to 48 hr, or isolated heart or embryonic explant culture for 48-72 hr. We also report an ultrasound-mediated microinjection protocol in mouse embryonic hearts in utero. This technique allows monitoring the development of embryos13 and allows for long-term follow-up of the injectates and/or labeled cells.
We found that these approaches preserve the function of the organ and provide a more representative environment than in vitro testing of stem cell potential. It also provides the opportunity to follow migration of labeled and/or injected cells to monitor their fate. Ultimately, this should yield a better understanding of regional tissue patterning and key biological processes.

<table border="0" cellpadding="0" cellspacing="0" width="986">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Setups / Hardware</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">40 MHz Transducer</td>
			<td style="width:175px;">VisualSonics</td>
			<td>MS550S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microinjector</td>
			<td style="width:175px;">VisualSonics</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td style="width:175px;">Eppendorf</td>
			<td>5242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator&#160;</td>
			<td style="width:175px;">Eppendorf</td>
			<td>5171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Nitrogen</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>required to pressurize the injector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rail system</td>
			<td style="width:175px;">VisualSonics</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">rotator</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>to rotate glass tube with embryos inside the incubator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Standard incubator</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>5% CO2, 37 C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stereomicroscope</td>
			<td style="width:175px;">Zeiss</td>
			<td style="width:104px;">Discovery. V8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Vevo 2100</td>
			<td style="width:175px;">VisualSonics</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microinjection</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">borosilicate capillary tubes</td>
			<td style="width:175px;">World Precision Instrument</td>
			<td style="width:104px;">KTW-120-6</td>
			<td>1.2 mm external diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pipette puller&#160;</td>
			<td style="width:175px;">Sutter</td>
			<td style="width:104px;">Model P87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">microinjection needles</td>
			<td>Origio-Humagen&#160;</td>
			<td>C060609</td>
			<td>OD/ID 1.14mm/53mm, with 50/35 um OD/ID tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringes&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Petridishes</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Silicon membrane</td>
			<td style="width:175px;">Visualsonics</td>
			<td style="width:104px;"></td>
			<td>4.3x4.3 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Play-Doh</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Isoflurane</td>
			<td style="width:175px;">Vet One</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hair removal agent&#160;</td>
			<td style="width:175px;">Nair</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:297px;">eye lubricant</td>
			<td style="width:175px;">Optixcare</td>
			<td>31779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Electrode gel (Signa)</td>
			<td style="width:175px;">Parker</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Suture</td>
			<td style="width:175px;">Sofsilk 5-0</td>
			<td style="width:104px;">S1173</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ultrasound gel</td>
			<td style="width:175px;">Aquasonic</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Buprenex Buprenex (buprenorphine hydrochloride)</td>
			<td style="width:175px;">Reckitt Benckiser Pharmaceuticals Inc.</td>
			<td style="width:104px;">NDC 12496-0757-1</td>
			<td>0.05-0.1 mg/kg in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 184&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glass petridishes</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:104px;"></td>
			<td>60mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">insect pins&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Media and culture reagents</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimem medium</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:104px;">51985026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M2 medium&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">M7167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Eagle Medium</td>
			<td style="width:175px;">Lonza</td>
			<td style="width:104px;">BE12-640F</td>
			<td>high glucose and 50% rat serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M16 medium&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">M7292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">rat serum</td>
			<td style="width:175px;">Janvier</td>
			<td style="width:104px;">ODI 7158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pennicilin/streptomycin&#160;</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:104px;">15140-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">oxygen 40%</td>
			<td style="width:175px;">Air liquid</td>
			<td style="width:104px;"></td>
			<td>required to oxygenate the embryo culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">fetal calf serum</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:104px;">RVJ35882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">matrigel</td>
			<td style="width:175px;">BD</td>
			<td style="width:104px;">356230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">collagen type I</td>
			<td style="width:175px;">BD</td>
			<td style="width:104px;">354236</td>
			<td>to coat culture dishes for explant culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">culture dishes</td>
			<td style="width:175px;">Dutcher /Orange</td>
			<td style="width:104px;">131020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Injectates</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDCFDA-SE&#160;</td>
			<td>Invitrogen/Molecular Probes&#160;</td>
			<td>C1165</td>
			<td>25mg/ml DMSO. Store at -20 C. Dilute 1:100-200 in saline before use.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PGK-GFP-expressing lentivirus&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>~8E9 transducing units/ml DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lipofectamine 2000&#160;</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:104px;">11668019</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell labeling and injection in developing embryonic mouse hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

Using the injection protocols described above, cells can be labeled and/or injected into the embryonic mouse heart. As proof of concept, several examples are shown in which the injection protocol and the ex vivo AVC explant, isolated heart, or whole embryo culture were combined (Figure 1).
Figure 1 shows the preparation of the embryo before cell injection. The E9.5 embryo is removed from its decidua while maintaining integrity of the yolk sac (<strong...

Watch this Scientific Journal Video about Cell Labeling and Injection in Developing Embryonic Mouse Hearts at JoVE.com

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

cell-labeling-and-injection-in-developing-embryonic-mouse-hearts

Research

JoVE Journal

Biology

14.0K Görüntüleme.  Aix-Marseille University.  Biz, (E) 9.5 ve daha sonraki aşamalarında embriyonik gün fare embriyoları olan embriyonik veya pluripotent hücrelerinden elde edilen iç ve aşılanmış hücrelerden hücre kaderine ve fenotipi hem izlenmesi amacıyla, boyalar enjekte DNA vektörleri, virüs, ve hücreler, bir dizi yöntem tarif gelişme. 