Name: the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development
Uploaded: 2016-10-05T15:00:00
Description: Watch this Scientific Journal Video about The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development at JoVE.com

The authors would like to acknowledge funding support for aspect of the work through Linkage Grants LP0776563 (GB, AS) and LP0211919 (GB) and industry partners Sappi and Mondi as well as Australian Postgraduate Award (EM) from the Australian Research Council and the Young Innovators and Scientist Award through the Australian Department of Agriculture (LT). We also like to thank the Zander Myburg, Qing Wang, Colleen MacMillan and Simon Southerton for the many discussions and ideas they put forward during the development of this protocol and to Martin Ranik, Minique De Castro, Julio Najera, Valerie Frassiant, Angelique Manuel and Noemie Defaix for assistance in laboratory related work.

1. Preparation of Plant Material
<ol>
	<li>Prior to experimentation, raise new seedlings of the preferred tree species from seed or cutting and grow tree/s until the diameter of the stem in the area intended for experimentation is approximately 1 cm in diameter. 
	Note: The time needed may vary due to plant growth rates therefore allow between three to nine months for this step.</li>
</ol>
2. Binary Vector Creation
<ol>
	<li>Conduct work from this section to section 7 in a laboratory or g...

<ol>
	<li>Spokevicius, A. V., Tibbits, J. F. G., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+plant+and+plant+part+transgenic+approaches+in+the+study+of+wood+formation+-+benefits+and+limitations.">Whole plant and plant part transgenic approaches in the study of wood formation - benefits and limitations.</a> TPJ. 1 (1), 49-59 (2007).</li><li>Chaffey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wood+formation+in+forest+trees:+from+Arabidopsis+to+Zinnia.">Wood formation in forest trees: from Arabidopsis to Zinnia.</a> Trends Plant Sci. 4 (6), 203-204 (1999).</li><li>Moller, R., McDonald, A. G., Walter, C., Harris, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+differentiation,+secondary+cell-wall+formation+and+transformation+of+callus+tissue+of+Pinus+radiata+D.+Don.">Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D. Don.</a> Planta. 217 (5), 736-747 (2003).</li><li>Spokevicius, A. V., Van Beveren, K., Leitch, M. M., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+in+vitro+transformation+of+wood-producing+stem+segments+in+eucalypts.">Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts.</a> Plant Cell Rep. 23 (9), 617-624 (2005).</li><li>Plasencia, 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=Eucalyptus+hairy+roots,+a+fast,+efficient+and+versatile+tool+to+explore+function+and+expression+of+genes+involved+in+wood+formation.">Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.</a> Plant Biotech J. , (2015).</li><li>Van Beveren, K. S., Spokevicius, A. V., Tibbits, J., Wang, Q., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+cambial+tissue+in+vivo+provides+efficient+means+for+Induced+Somatic+Sector+Analysis+(ISSA)+and+gene+testing+in+stems+of+woody+plants+species.">Transformation of cambial tissue in vivo provides efficient means for Induced Somatic Sector Analysis (ISSA) and gene testing in stems of woody plants species.</a> Funct Plant Biol. 33 (7), 629-638 (2006).</li><li>Spokevicius, A. V., Van Beveren, K., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+transformation+of+dormant+lateral+buds+in+poplar+trees+reveals+developmental+patterns+in+secondary+stem+tissues.">Agrobacterium-mediated transformation of dormant lateral buds in poplar trees reveals developmental patterns in secondary stem tissues.</a> Funct Plant Biol. 33 (2), 133-139 (2006).</li><li>Spokevicius, A. V., 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=beta-tubulin+affects+cellulose+microfibril+orientation+in+plant+secondary+fiber+cell+walls.">beta-tubulin affects cellulose microfibril orientation in plant secondary fiber cell walls.</a> Plant J. 51 (4), 717-726 (2007).</li><li>MacMillan, C. P., 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=The+fasciclin-like+arabinogalactan+protein+family+of+Eucalyptus+grandis+contains+members+that+impact+wood+biology+and+biomechanics.">The fasciclin-like arabinogalactan protein family of Eucalyptus grandis contains members that impact wood biology and biomechanics.</a> New Phytol. 206 (4), 1314-1327 (2015).</li><li>Creux, N. M., Bossinger, G., Myburg, A. A., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+somatic+sector+analysis+of+cellulose+synthase+(CesA)+promoter+regions+in+woody+stem+tissues.">Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues.</a> Planta. 237 (3), 799-812 (2013).</li><li>Hussey, S. G., 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=SND2,+a+NAC+transcription+factor+gene,+regulates+genes+involved+in+secondary+cell+wall+development+in+Arabidopsis+fibers+and+increases+fiber+cell+area+in+Eucalyptus.">SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibers and increases fiber cell area in Eucalyptus.</a> BMC Plant Biology. 11, (2011).</li><li>Melder, E., Bossinger, G., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+ARBORKNOX1+delays+the+differentiation+of+induced+somatic+sector+analysis+(ISSA)+derived+xylem+fiber+cells+in+poplar+stems.">Overexpression of ARBORKNOX1 delays the differentiation of induced somatic sector analysis (ISSA) derived xylem fiber cells in poplar stems.</a> Tree Genet. Genomes. 11 (5), (2015).</li><li>Baldacci-Cresp, F., 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=PtaRHE1,+a+Populus+tremula+x+Populus+alba+RING-H2+protein+of+the+ATL+family,+has+a+regulatory+role+in+secondary+phloem+fiber+development.">PtaRHE1, a Populus tremula x Populus alba RING-H2 protein of the ATL family, has a regulatory role in secondary phloem fiber development.</a> Plant J. 82 (6), 978-990 (2015).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. , (2001).</li><li>Murashige, T., Skoog, 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+revised+medium+for+rapid+growth+and+bio-assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio-assays with tobacco tissue cultures.</a> Physiol. Plant. 15 (3), 473-497 (1962).</li><li>Hawkins, S., Pilate, G., Duverger, E., Boudet, A., Grima-Pettenati, J., Chaffey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+GUS+histochemistry+to+visualise+lignification+gene+expression+in+situ+during+wood+formation.">The use of GUS histochemistry to visualise lignification gene expression in situ during wood formation.</a> Wood formation in trees: Cell and Molecular Biology Techniques. , 271-295 (2002).</li><li>Hodal, L., Bochardt, A., Nielsen, J. E., Mattsson, O., Okkels, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+expression+and+specific+elimination+of+endogenous+beta-glucuronidase+activity+in+transgenic+and+nontransgenic+plants.">Detection, expression and specific elimination of endogenous beta-glucuronidase activity in transgenic and nontransgenic plants.</a> Plant Sci. 87 (1), 115-122 (1992).</li><li>Hansch, R., Koprek, T., Mendel, R. R., Schulze, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+for+eliminating+endogenous+beta-glucuronidase+background+in+barley.">An improved protocol for eliminating endogenous beta-glucuronidase background in barley.</a> Plant Sci. 105 (1), 63-69 (1995).</li></ol>

The ISSA protocol is a relatively simple and efficient method for the creation of transgenic stem tissues in tree species in the space of a few months for analysis of genes and promoters of interest involved in wood and stem formation. Little effort, beyond keeping plants alive, is required to grow transgenic stem tissue following inoculation which stands in contrast to in vitro methods where extensive culturing is required to maintain tissue or plants, where wood production can take up to years to commence or w...

Tree stems comprise a significant amount of the planets biomass and are of immense biological, cultural and commercial importance. Secondary stems create habitat by providing resources and shelter for many other life forms. They deliver many other services to the ecosystems they inhabit and act as a renewable resource for the production of timber, pulp and paper and other wood and non-wood products. Secondary stem development and more specifically wood formation is governed by complex molecular system that regulate the development of specific cell types, the biochemical composition of their cell walls and how they are arranged to form tissues and organs. Dissecting the molecular basis of secondary stem development and wood formation is confounded by many factors including the variability of wood and stem properties within and between stems, long generation times, out-crossing mating systems, high heterozygosity, high genetic load, seasonal dormancy, long mature trait establishment periods and the sheer physical size of mature trees. As a result, understanding of secondary stem development relative to the detailed knowledge of most other aspects of the molecular control of plant development, is still in its infancy.
A number of in vitro techniques have been used to study and understand secondary stem development, in particular wood and secondary cell wall formation. These protocols involve the use of whole plant or plant part systems, where either transgenic plants are created or specific secondary cells or tissue are transformed for the study of specific aspects of wood and/or secondary stem development1. Transgenic plants can be recovered post genetic transformation from a wide variety of plant tissues and cell types, however, progress is slow, particularly when analyzing wood fiber traits due to long regeneration and stem maturation times (in the order of years), high technical and labor demands, low throughput of candidate genes as well as difficulties in propagating some woody plant species. Similar techniques have been developed in non-woody model system such as Arabidopsis that successfully overcome some of these limitations, but not all secondary stem cell types a present in these stems and traits related to seasonality or longevity cannot be studied in such species2. Alternatively, plant part systems, such as Pinus radiata callus cultures3 reduce the associated timeframes. These methods however are restricted to the study of an individual cell type and suffer similar constraints as noted for in vitro experiments. Similarly, apical stem cultures4 involving whole stem explants have shown promise but as yet have not been applied for the study of specific genes or promoters of interest. More recently, an alternative protocol involving hairy root cultures has been developed for eucalypts and has been successfully applied5, however, this method still requires in vitro cultivation, involves secondary roots rather than stems and to date it is limited to a single tree species.
Induced somatic sector analysis (ISSA), as described here, was developed to overcome some of these problems providing a medium to high throughput functional screening tool for genes and promoters with suspected roles in wood formation and secondary stem tissue development. ISSA is an in vivo transformation and screening system which was developed to reduce the time taken to produce transgenic cells and tissue in an intact secondary stem while overcoming labor, technical and throughput limitations of routinely used in vitro methods. The protocols described here allow for the simultaneous creation of hundreds of independently transformed tissue sectors and cells in secondary stems within a short period of time, in the tree species and tissue of interest without genetic and/or environmental variation within relatively short time frames and low labor cost. ISSA in vivo techniques were first described for secondary stem6 and bud7 tissues and have since been refined in secondary stem tissue through studies of genes and/or promoters involved in cambial differentiation and include: tubulin (TUB)8, fasciclin-like arabinogalactan (FLA)9, cellulose synthase (CESA)10, secondary cell wall-associated nac domain (SND2)11, ARBORKNOX (ARK1)12 and really interesting new gene (RING) H2 protein13. These studies were conducted in secondary stems of poplar and eucalypt plants and provided insights into cell morphology, cell wall chemistry and gene expression.
The protocols described here are intended to bring together the experience and knowledge gained through the development and application of ISSA from a range of published and unpublished studies over the last decade. They focus on the in vivo transformation of secondary stem tissues6 and concentrate on studies involving Populus alba &#39;pyramidalis&#39; clone, Eucalyptus globulus as well as 11 Eucalyptus globulus x camaldulensis clones. This document takes researchers through the protocol from the cultivation of plants and bacteria, transformation of stem tissues, growth and harvest of tissue, identification of transgenic cells and tissues, preparation for phenotypic assessments and methods for the collection and analysis of data. While techniques have successfully been applied to measure cell wall monosaccharide composition also9,11, due to space limitations, this document concentrates on techniques used for measuring cell and tissue morphology and understanding gene expression patterns in secondary stems only. Accordingly, the protocol as outlined is suited to those looking to gain further insights into the role and/or expression of genes linked to secondary stems using a low cost, technically easy, and medium to high throughput method.

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Plants</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">Please consult local nursery suppliers for plants as needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Agrobacterium strain</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">There are many possible avenues to obtain Agrobactrium strains. We suggest you follow up within your local research community as there may be restrictions in obtaining the bacteria in your country and region.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:188px;">Binary vector (gene and promoter)</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">We have developed a range of vectors to suite the ISSA protocol using a the Gateway Recombinase system. This include overexpression, RNAi knockouts and promoter fusion vectors based on modified pCAMBIA vectors and happy to provide as needed. In addition, there are many vectors avialable to the research community.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LB media</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">L3022</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LB media with agar</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">L2897</td>
			<td style="width:751px;">A like product could be sourced from another company</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Antibiotics</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">The catalog number will be dependent on the antibiotic you require as a range of antibiotic are used for bacterial selection in binary vectors. This product could be sourced from a&#160; range of companies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">50 ml Screw top tubes</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">2 ml Microtube</td>
			<td style="width:95px;">Watson Bio Lab</td>
			<td style="width:119px;">132-620C</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:188px;">MS Media</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">M9274</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Scalpel blade no 11</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">S2771</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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			<td height="21" style="height:21px;width:188px;">Parafilm &#34;M&#34;</td>
			<td style="width:95px;">Bemis</td>
			<td style="width:119px;">PM996</td>
			<td style="width:751px;">This is the best product to use to bind the cambial window post creation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">14 ml round bottom tubes</td>
			<td style="width:95px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">150268</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">EDTA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Triton</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:188px;">X-Gluc</td>
			<td style="width:95px;">X-GLUC direct</td>
			<td style="width:119px;"></td>
			<td style="width:751px;">You will need to go to the website to order - http://www.x-gluc.com/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Potassium Ferricyanide (III)</td>
			<td style="width:95px;">Sigma</td>
			<td align="right" style="width:119px;">244023</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Potassium Ferrocyanide (II)</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">P9387</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Litmus paper</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">WHA10360300</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Single edge razor blade</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L055</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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			<td height="21" style="height:21px;width:188px;">Double edge razor blade</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L056</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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			<td height="21" style="height:21px;width:188px;">SEM Pin Stub</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">GTP16111</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Sample vial with screw cap</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L6204</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Ethanol</td>
			<td style="width:95px;">sigma</td>
			<td style="width:119px;">E7023</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LR white</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">C025</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Embedding Mould</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">RL090</td>
			<td>We recommend this variety, however there are plenty of options available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Water Soulable mounting media</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">IA019</td>
			<td>One example of a mounting media that could be used however other options do exist and could be explored.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Hydrogen peroxide</td>
			<td style="width:95px;">Sigma</td>
			<td align="right" style="width:119px;">216763</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Glacial acetic acid</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">A9967</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Safranin O</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">C138</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:188px;">Quanta Environmental Scanning Electron Microscope</td>
			<td style="width:95px;">FEI</td>
			<td style="width:119px;"></td>
			<td>This is the instrument used at part of this study but any other SEM that has a low vacuum mode could be utilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Image J imaging software</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;can be sourced from the following URL http://rsbweb.nih.gov/ij/</td>
		</tr>
	</tbody>
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the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

Using this protocol all live secondary stem cell and tissue types have been shown to be susceptible to A. tumefaciens transformations and have been defined into sector types based on the cell type initially transformed and it subsequent developmental growth pattern. Sector types include periderm, phloem, cambial, wound parenchyma and tylose (Figure 1b, 1c, 1d) and can be found in consistent locations describe in the remainder of ...

Watch this Scientific Journal Video about The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development at JoVE.com

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

The overall goal of Induced Somatic Sector Analysis is to provide a medium to high throughput technique for the functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. This method can help answer key questions around where and when danger expressed as well as their role in biologically important processes such as wood formation and secondary stem development. The main advantage of this technique is that it is efficient, easy and relatively a fast procedure that is applicable to a wide range of tree species.To begin, prepare a clean, 50 milliliter screw cap tube with 19 milliliters of fresh, 28 degree Celsius LB medium with the appropriate antibiotics for bacterial selection. Add one milliliter of A.tumefaciens culture and check the optical density, or OD, at 600 nanometres. If the OD is above 0.1, dilute further with warm LB.Place the A.tumefaciens culture into a shaking incubator until the OD 600 is between 0.4 and 0.6.Once this is achieved, centrifuge the cells for 15 minutes at four degrees Celsius and 1, 000 x g. Decant the liquid from the tube and immediately re-suspend the cell pellet with one milliliter of cooled MS medium. Transfer the suspension to a clean, two milliliter microtube and store on ice until required.Begin the experiment during the spring or early summer and select plants with an active and fast growing cambium. This is confirmed if the phloem can be easily peeled from the xylem. Identify a straight section of stem near the base of the plant and remove any leaves or branches.Using a sharp scalpel, cut a one square centimeter cambial window by creating two parallel, 20 millimeter incisions through the phloem at five millimeters apart and then a single, horizontal cut that bisects the two vertical cuts at the basal end. Heal the phloem strip upwards exposing the developing xylem tissue. And then, using a pipette, add sufficient inoculation medium to fully wet the surface of the exposed xylem.Immediately, reinsert the phloem strip into the stem and bind it into place tightly with parafilm. Repeat these inoculation steps for all genes or promoters of interest creating new cambial windows at least one centimeter above or below existing windows and at a 90 degree offset. Additionally, inoculate windows with the positive and negative control vectors on the same stem or stems, as the experimental inoculates.Monitor stem growth periodically and once radial growth of at least five millimeters has been observed, harvest the tissue for a beta-glucuronidase, or GUS assay. To harvest the test material, first excise the cambial window from the stem removing any tissue that is not part of new growth. For studies relating to mature xylem and phloem cell or tissue morphology, peel the phloem from the xylem.Alternatively, for investigations requiring that the cambial zone remain intact, or promoter expression patterns to be assessed, slice the cambial window transversely using a razor blade to create discs of between 0.5 and one millimeter thickness. Place the harvested cambial window tissues into individual, 14 milliliter round bottom tubes and rinse twice in 0.1 molar phosphate buffer at pH 7 for five minutes each time ensuring that the tissue is completely submerged. At the end of the second rinse, take care to remove all excess solution.Next, add five milliliters of GUS reagent to each tube. If this does not completely cover the tissue sample, add additional reagent. Incubate the tubes for ten minutes at 55 degrees Celsius in the dark.Following this, incubate the tubes for a further 12 to 16 hours on a shaker at 37 degrees Celsius in the dark using gentle agitation. Upon removal from the incubator, take a random subset of the tubes and check the pH of the GUS reagent using litmus paper with pH range of zero to seven. If any of them have a pH lower than six, label them and check the remainder of the tubes.Label and exclude any tubes with a pH lower than six. Decant the GUS reagent from the tubes and replace it with sufficient 70 percent ethanol to cover the tissue. Store the samples at four degrees Celsius until needed.Using a clean pair of forceps, remove all the cambial window tissue from a sample tube and place it onto a small tray or petri dish for microscopic visualization. Place the sample under the lens of a dissecting microscope between 1X and 4X magnification and identify and tally the number of cells or tissues displaying bright blue staining. In examples where the phloem was removed, count the number of sectors in cambial tissue found on the surface of the developing xylem.For samples cut into discs, count the number of sectors found in the different cell or tissue types. Ensure that both sides of the disc are viewed and that sectors that occur across two discs are matched to ensure that numbers are not overestimated. Place only the tissue containing sectors back into the tube containing 70 percent ethanol and store until needed.Repeat the analysis for any additional cambial windows harvested including the positive and negative controls. To aid in the accurate identification in the classification of sectors, we suggest you become familiar with secondary stem anatomy and development including stem responses to wounding. This figure shows several successful transformations seen here as regions of blue staining.An example phloem peel window displays multiple transformed sectors and discs harvested from two tree species show sectors of transformation and subsequent radial growth. Transformation in a range of tissue types can also be observed. Here stem sectors can be seen in the periderm, phloem, wound parenchyma, and several other tissue regions of white poplar.This image shows the results of gene promoter expression in cambial derivatives. In this example, we see the proportion of staining in each tissue type in a trial involving a range of CesA promoters from eucalyptus grandis transformed into stems of eucalypt hybrids. Expression in the phloem, developing and developed xylems is represented as a percentage.Average number of transformation events per centimeter of cambium inoculated can also be calculated for each promoter or gene of interest. This table shows the data for typical sectors identified in eucalypt and poplar subjects transformed with a positive control or promoter of interest analyzed using the disc harvesting method. Once mastered, this technique can provide a fast, efficient and widely applicable method for dissecting the complex nature of cambial differentiation with formation in secondary stem development.After watching this video, you should have a good understanding of how to prepare plants and bacteria, how to transform, grow and harvest stems as well as how to identify and classify transgenic tissue for phenotypic assessment.

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Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.