The authors thank Ms. Jennifer M. Lommel and Tara N. Rognan for maintenance of Arabidopsis plants. This work was supported by start-up funds from Saint Louis University and National Institutes of Health grants 1R15GM086846-01 and 3R15GM086846-01S1 to W. Xiao.

I. Genetic Crossing
1. Emasculating the female parent
In order to distinguish the maternal and paternal alleles by using DNA sequence polymorphism, two different ecotypes, e.g. Columbia-0 (Col-0) and Ler, will be chosen as female and male parents. The plants should be young and healthy. One can emasculate the female parent by using a dissecting microscope, magnifying visor, or naked eye. Locate stage-12 flowers (Smyth et al., 1990) and remove any flowers or si...

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. 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+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+sodium+bisulfite+sequencing+in+plants.">Accurate sodium bisulfite sequencing in plants.</a> Epigenetics. 5, 47-49 (2010).</li><li>Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+demethylation+of+Arabidopsis+endosperm.">Genome-wide demethylation of Arabidopsis endosperm.</a> Science. 324, 1451-1454 (2009).</li><li>Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+hypermethylation+of+flower-specific+genes+in+Arabidopsis.">Ectopic hypermethylation of flower-specific genes in Arabidopsis.</a> Curr. Biol. 10, 179-186 (2000).</li><li>Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-way+control+of+FWA+imprinting+in+Arabidopsis+endosperm+by+DNA+methylation.">One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.</a> Science. 303, 521-523 (2004).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+integrated+single-base+resolution+maps+of+the+epigenome+in+Arabidopsis.">Highly integrated single-base resolution maps of the epigenome in Arabidopsis.</a> Cell. 133, 523-536 (2008).</li><li>Lister, R., Pelizzola, M., Dowen, R. H., Hawkins, R. D., Hon, G., Tonti-Filippini, J., Nery, J. R., Lee, L., Ye, Z., Ngo, Q. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462, 315-322 (2009).</li><li>Paulin, R., Grigg, G. W., Davey, M. W., Piper, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urea+improves+efficiency+of+bisulfite-mediated+sequencing+of+5'-+methylcytosine+in+genomic+DNA.">Urea improves efficiency of bisulfite-mediated sequencing of 5'- methylcytosine in genomic DNA.</a> Nucl. Acids Res. 26, 5009-5010 (1998).</li><li>Rogers, S. O., &amp;amp, B. e. n. d. i. c. h., J, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+plant+tissues.">Extraction of DNA from plant tissues.</a> Plant Molecular Biology Manual. A6, 1-10 (1988).</li><li>Smyth, D. R., Bowman, J. L., Elliot, M., Meyerowitz, E. 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+Flower+Development+in+Arab&#237;dopsis.">Early Flower Development in Arab&#237;dopsis.</a> Plant Cell. 2, 755-767 (1990).</li><li>Xiao, W., Gehring, M., Choi, Y., Margossian, L., Pu, H., Harada, J. J., Goldberg, R. B., Pennell, R. I., Fischer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+MEA+Polycomb+gene+is+controlled+by+antagonism+between+MET1+methyltransferase+and+DME+glycosylase.">Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase.</a> Dev. Cell. 5, 891-901 (2003).</li></ol>

It is relatively easy to separate embryo from endosperm and seed coat, but it is tedious to separate endosperm from seed coat, especially for seed at early or mid-torpedo stage of embryogenesis. Since seed coat only contributes a very small amount of tissue, for some genes, e.g., MEA and FWA, we do not have to separate endosperm from seed coat. That means that we can isolate RNAs from a mixture of endosperm and seed coat tissues, check expression of the maternal and paternal alleles of a gene, compare w...

Supplies
<ul>
 <li>Dissecting Microscope</li>
 <li>Scissors</li>
 <li>Fine Tip Forceps</li>
 <li>Jewelry Tag</li>
 <li>Plant Stakes</li>
 <li>String or Twist-Ties</li>
 <li>4" X 2" X 8" Polyethylene Bags</li>
 <li>3" X 1" X 1.0 mm Microscope Slides</li>
 <li>Liquid Nitrogen</li>
 <li>Liquid Nitrogen Containers</li>
 <li>Heat Block</li>
 <li>PCR Tubes</li>
 <li>Thermocycler</li>
 <li>Microcentrifuge Tubes</li>
 <li>Microcentrifuge</li>
 <li>Gel Electrophoresis facility</li>
 <li>Arabidopsis thaliana Columbia-0 Plants</li>
 <li>Arabidopsis thaliana Landsberg erecta Plants</li>
</ul>
Reagents
<ul>
 <li>70% Ethanol</li>
 <li>95% Ethanol</li>
 <li>100% Ethanol</li>
 <li>0.3 M Sorbitol and 5 mM MES-pH 5.7</li>
 <li>Cetyltrimethyl Ammonium Bromide (CTAB)</li>
 <li>100% Ethanol</li>
 <li>Cholorform</li>
 <li>Restriction Enzymes</li>
 <li>3 M NaOH</li>
 <li>6.3 M NaOH</li>
 <li>6.24 M Urea/ 4 M Sodium Bisulfite</li>
 <li>Sterile distilled H2O</li>
 <li>10 mM Hydroquinone</li>
 <li>Wizard DNA Clean-Up System (Promega)</li>
 <li>10 M NH4OAc</li>
 <li>20 &mu;g/ &mu;L tRNA</li>
 <li>TE buffer</li>
 <li>The TOPO TA Cloning Kit (Invitrogen)</li>
</ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

determination-dna-methylation-imprinted-genes-arabidopsis

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Biology

17.3K visualizações.  Saint Louis University. Imprinting é um fenômeno em plantas e da reprodução de mamíferos. Metilação do DNA desempenha um papel importante nos mecanismos de imprinting. Isolando endosperma e determinação do estatuto de metilação de genes imprinted em Arabidopsis Pode ser difícil. Neste protocolo, descrevemos como isolar endosperma e determinar a metilação por seqüenciamento bissulfito.