Name: genomic transformation of the picoeukaryote ostreococcus tauri
Uploaded: 2012-07-13T13:00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Genomic Transformation of the Picoeukaryote Ostreococcus tauri at JoVE.com

SynthSys a Centre for Integrative Systems Biology funded by BBSRC and EPSRC award D019621. EU FP6 Network of Excellence "Marine Genomics" grant to FYB has financed development of genetic transformation methods.

1. Preparation of Algal Material
<ol>
	<li>Ostreococcus cells are cultured in artificial seawater (ASW). Sea salts (typically about 40 grams per liter) are dissolved in deionised water to a salinity of 30 ppt, as measured using a salinity meter. Enrichment medium11,12, trace metal elements and vitamins are added as described in Tables 1-3. The media is then filter-sterilised through a 0.22 &mu;m filter.</li>
	<li>For maintenance, cells of Ostreococcus strain OTTH95 are sub-cultured aseptically at a dilution of 1 /100 in fresh ASW every 7 days and grown under constant light in a plant growth incubator fitted with Moonlight Blue filter. The light intensity should be close to 20 &mu;mol m-2 s-1 and temperature is maintained at 20 &deg;C. Cells do not require constant agitation, but are shaken once every 2 to 3 days to prevent aggregation.</li>
	<li>For each transformation, 50 ml of cells are required at a cell density of 20-30 x 106 ml-1, which should be attained 5-7 days following sub-culturing. Approximate cell density and axeny can be determined using either flow cytometry or a haemocytometer at a minimum of x40 magnification.</li>
</ol>
2. Electroporation
<ol>
	<li>Prepare DNA for the transformation. For each transformation, 5 &mu;g of pure, linearised plasmid DNA is required at a concentration of 1 &mu;g/&mu;l in sterile deionised water. To obtain this DNA, the authors recommend using a Qiagen midi prep kit, though other methods might work equally well. Digest the product with an enzyme that cuts in the backbone of the vector used, but not in the transgene or selection gene. Further purify and concentrate the resulting linear DNA by ethanol precipitation, and resuspend the product in the appropriate volume of high-quality sterile deionised water.</li>
	<li>Prepare microcentrifuge tubes containing 5 &mu;g DNA for each transformation. A control with no DNA is necessary for each cell line to be transformed. Keep these tubes on ice, together with a 2 mm electroporation cuvette for each transformation.</li>
	<li>Prepare 2.2 ml of resuspension buffer per transformation. Dissolve Sorbitol to a 1 M solution in ddH2O, add 0.1 % pluronic acid F68 and filter-sterilise.</li>
	<li>Add pluronic acid F68, to a final concentration of 0.1% to the cells, and pellet them for 10 minutes at 8000x g at 10 &deg;C in a 50 ml tube with conical bottom. Immediately resuspend the cells in 1 ml of resuspension buffer by pipetting up and down, and transfer to a microfuge tube. Spin down for 10 minutes at 8000 x g at 10 &deg;C, and working quickly, repeat this wash step once more.</li>
	<li>With a cut tip, resuspend each final pellet in 40 &mu;l of resuspension buffer. Add 40 &mu;l of the resuspended cells to every tube of linearised DNA on ice, mix gently and transfer to the electroporation cuvette.</li>
	<li>Put the cuvette in the electroporation machine. Change the settings to 6 kV cm-1, 600 &Omega;, and 25 &mu;F. Electroporate the cells.</li>
	<li>Incubate the electoporated cells in the cuvettes at room temperature for 10 minutes, and use that time to prepare the tissue culture flasks. Label them and add 30 ml of fresh ASW to each. Take 1 ml out of each flask and gently add it to the corresponding cuvette. Incubate for 2 minutes and gently remove the ASW, now containing the globule of cells and gently and slowly pipette directly into the ASW in the culture flask.</li>
	<li>Cells typically remain in a globule. Take care not to shake or disturb the aggregated cells at this moment. Allow the cells to recover in the incubator for 1-2 hours, then resuspend by shaking the flasks. At this time, the cells should resuspend freely and no clumps should be visible. Leave the cultures to recover overnight in the incubator.</li>
</ol>
3. Inclusion of Cells on Plates in Semi-solid Medium
<ol>
	<li>The next day, autoclave a solution of 2.1% low melting point agarose in ddH2O in a bottle containing a stirring rod. Keep molten at 65-90 &deg;C in a larger beaker containing water on a heating magnetic stirrer. For each transformation prepare 8 Petri dishes (55 mm diameter) and 8 x 15 ml tubes each containing 9 ml of ASW plus the required selection. If using Nourseothricin or G418, use 2 mg/ml.</li>
	<li>Collect the transformed cells from the incubator. Working in a sterile flowhood, add 1 ml of boiling LMP agarose to the 9 ml in one of the tubes. Close the tube, and mix by inverting. Then add 0.5 ml of freshly transformed cells, quickly mix and pour into the plate. Repeat this process with the remaining 7 tubes and then proceed to the next transformation flask.</li>
	<li>Leave the plates open in the flow hood for an hour, for the agarose to set. Then close the plates and transfer them to large square Petri dishes, which will hold 4 plates each. Seal the square plates with parafilm. Note that the plates will not set completely, and as a result, the gel is very fragile. Care should be taken not to break the gel when handling the plates. Place all square plates in the incubator.</li>
</ol>
4. Selection of Transformed Colonies
<ol>
	<li>Colonies should appear after 10 to 21 days on the transformation plates, but not on the mock transformed plates. To pick colonies use a 200 &mu;l pipette with cut-off tips. Simply select free colonies and suck out the green colony. Take care not to include any cells from neighbouring colonies.</li>
	<li>Transfer the cells to 2 ml of liquid medium containing the selection, in a 24 wells plate. When using Nourseothricin, use 1.75 mg/ml. Mix by pipetting up and down. Select 24-50 colonies per transformation. Seal the plates with parafilm and transfer to the incubator.</li>
	<li>After a week, transfer 100 &mu;l of each well to 2 ml fresh ASW with selection in a 24 wells plate and grow for an additional 7 days. Surviving cell lines can be used for further studies. Stable integration into the genome and insertion number should be analysed using PCR and Southern Blot. Genomic DNA can be obtained for these purposes using any down-scaled standard method for plant DNA extraction.</li>
</ol>
5. Representative Results
Cells split 1 / 100 reached a density of 25 million cells per milliliter after growing for 7 days. Electroporation of the cells with the appropriate settings resulted in a time-constant between 10 and 14 milliseconds. When cells are transferred to medium in culture flasks, a globule of cells should form. When lightly shaken after an hour, the cells should easily resuspend. Inclusion of 1 ml transformed cells into 0.2% LMP agar should result in a consistent but only semi-solid gel. Colonies should appear after 10 to 21 days. When transforming 5 &mu;g of linearised DNA using the protocol above, 50-100 colonies per transformation plate were typically expected, versus none on the negative control plates. ~80% of colonies picked were positively selected by antibiotic resistance in liquid medium and were used in subsequent studies.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Final concentration</td>
			<td>Stock solution</td>
			<td>Volume for 1 liter</td>
		</tr>
		<tr>
			<td>NaNO3</td>
			<td>8.83 x 10-4 M</td>
			<td>75 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>3.63 x 10-5 M</td>
			<td>2.68 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>&beta;-glycerophosphate</td>
			<td>1 x 10-5 M</td>
			<td>2.16 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>H2SeO3</td>
			<td>1 x 10-8 M</td>
			<td>1.29 mg/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Tris-base(pH 7.2)</td>
			<td>1 x 10-3 M</td>
			<td>121.1 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>K trace metal solution</td>
			<td>&nbsp;</td>
			<td>Table 2</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>f/2 vitamin solution</td>
			<td>&nbsp;</td>
			<td>Table 3</td>
			<td>0.5 mL</td>
		</tr>
	</tbody>
</table>
Table 1. Medium constituents for growth of O. tauri11, 12. Make up a total volume of 1 liter of artificial seawater to a salinity of 30 ppt, and add the components above from stock solutions as indicated. Filter-sterilise the medium and use within a week. Stocks of all compounds except the vitamin solution can be pooled to add 6 ml of this solution for every liter of medium.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Final concentration</td>
			<td>Stock solution</td>
			<td>Amount for 1 liter</td>
		</tr>
		<tr>
			<td>Na2EDTA &bull; 2H2O</td>
			<td>1 x 10-4 M</td>
			<td>&nbsp;</td>
			<td>41.6 g</td>
		</tr>
		<tr>
			<td>FeCl3 &bull; 6H2O</td>
			<td>1 x 10-5 M</td>
			<td>&nbsp;</td>
			<td>3.15 g</td>
		</tr>
		<tr>
			<td>Na2MoO4 &bull; 2H2O</td>
			<td>1 x 10-8 M</td>
			<td>6.3 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>ZnSO4 &bull; 7H2O</td>
			<td>1 x 10-9 M</td>
			<td>22.0 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>CoCl2&bull; 6H2O</td>
			<td>1 x 10-9 M</td>
			<td>10.0 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>MnCl2 &bull; 4H2O</td>
			<td>1 x 10-8 M</td>
			<td>180.0 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>CuSO4&bull; 5H2O</td>
			<td>1 x 10-8 M</td>
			<td>9.8 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
	</tbody>
</table>
Table 2. Trace metal solution11,12. Make up to a total of 1 liter, in dH2O, and heat to dissolve. Aliquot the solution and freeze for storage. Final concentrations indicated refer to the final medium, not the trace metal solution.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Final concentration</td>
			<td>Stock solution</td>
			<td>Amount for 1 liter</td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>1 x 10-10 M</td>
			<td>1 g/L dH2O</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>1 x 10-9 M</td>
			<td>0.1 g/L dH2O</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Thiamine &bull; HCl</td>
			<td>1 x 10-7 M</td>
			<td>&nbsp;</td>
			<td>200 mg</td>
		</tr>
	</tbody>
</table>
Table 3. f/2 vitamin solution11. Make up to a total of 1 liter in dH2O, filter-sterilise, aliquot and freeze. Final concentrations indicated refer to the final medium, not the vitamin solution.
<img alt="Figure 1" src="/files/ftp_upload/4074/4074fig1.jpg" /> 
Figure 1. Graphical overview of the transformation procedure. Schematic representation of the procedure to genetically transform Ostreococcus tauri by electroporation.
<img alt="Figure 2" src="/files/ftp_upload/4074/4074fig2.jpg" /> 
Figure 2. Culture growth. Cells are sub-cultured aseptically at a dilution of 1 /100 in fresh ASW every 7 days and grown under constant light in a plant growth incubator fitted with Moonlight Blue filter. The light intensity should be close to 20 &mu;mol m-2s-1 and temperature is maintained at 20 &deg;C Cells do not require constant agitation, but are shaken once every 2 to 3 days to prevent aggregation.
<img alt="Figure 3" src="/files/ftp_upload/4074/4074fig3.jpg" /> 
Figure 3. Colony formation on 0.2% LMP agarose. Transfer 4 plates to a large square Petri dish, and seal with parafilm (top left). When colonies have formed, simply select free (ideally conically shaped) colonies (top right) and suck them out of the plate with a p200 pipette. There should be no colonies on the negative control (bottom right).

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+persist+without+transcription+in+a+eukaryote.">Circadian rhythms persist without transcription in a eukaryote.</a> Nature. 469, 554-558 (2011).</li><li>van Ooijen, G., Dixon, L. E., Troein, C., Millar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+function+is+required+for+biological+timing+throughout+the+twenty-four+hour+cycle.">Proteasome function is required for biological timing throughout the twenty-four hour cycle.</a> Curr. Biol. 21, 869-875 (2011).</li><li>Troein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+light+inputs+to+a+simple+clock+circuit+allow+complex+biological+rhythms.">Multiple light inputs to a simple clock circuit allow complex biological rhythms.</a> Plant. J. 66, 375-385 (2011).</li><li>Corellou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clocks+in+the+green+lineage:+comparative+functional+analysis+of+the+circadian+architecture+of+the+picoeukaryote+ostreococcus.">Clocks in the green lineage: comparative functional analysis of the circadian architecture of the picoeukaryote ostreococcus.</a> Plant Cell. 21, 3436-3449 (2009).</li><li>O'Neill, J. S., Reddy, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clocks+in+human+red+blood+cells.">Circadian clocks in human red blood cells.</a> Nature. 469, 498-503 (2011).</li><li>Monnier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrated+transcription+of+biological+processes+in+the+marine+picoeukaryote+Ostreococcus+exposed+to+light/dark+cycles.">Orchestrated transcription of biological processes in the marine picoeukaryote Ostreococcus exposed to light/dark cycles.</a> B.M.C. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+marine+planktonic+diatoms.+I.+Cyclotella+nana+Hustedt,+and+Detonula+confervacea+(cleve)+Gran.">Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran.</a> Can. J. Microbiol. 8, 229-239 (1962).</li><li>Keller, M. D., Selvin, R. C., Claus, W., Guillard, R. 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=Media+for+the+culture+of+oceanic+ultraphytoplankton.">Media for the culture of oceanic ultraphytoplankton.</a> J. Phycol. 23, 633-638 (1987).</li><li>Djouani-Tari, e. l. 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No conflicts of interest declared.

Cellular state and culture axeny is of crucial importance for the transformation procedure. Although cells are usually maintained in cycles of 12 hours light, 12 hour dark, transformation efficiency in cells grown under these conditions were found to be insufficient. During the repeated pelleting / resuspension steps, cells should always resuspend easily. If cells aggregate, or a mucus-like substance is present, it is better to start the procedure from the beginning again. Typically, ~50 colonies can be expected on each transformation plate, versus none on the negative control plate. In case of low transformation efficiency, culturing conditions should be varied to ensure cells are in the optimal state. Variables that affect transformation efficiency include ambient temperature in the laboratory (should be close to 20 &deg;C) and handling speed (Procedure from pelleting cells [2.3] to recovery [2.7] should take ~1 hour). It is also important that all solutions are made fresh before each transformation. For inclusion in plates, the concentration of LMP agarose is crucial. Ostreococcus cells will not grow in high agarose concentrations, and will diffuse in low concentrations.
Three transformation vectors for Ostreococcus have been published previously6, and more vectors are expected to be generated and published shortly. The existing vector Pot-Luc6 allows the use of a promotor of choice in combination with a C-terminal firefly luciferase tag allowing faster transgenic line selection plus tractable expression patterns, whereas the Potox vector6 carries the strong inducible13 promotor taken from the Ostreococcus High Affinity Phosphate Transporter (HAPT) gene for overexpression of the gene of interest. The Potox-Luc vector derives from Potox, carrying an additional luciferase marker that can be used for indirect selection of overexpression lines14. Successful selection markers on these vectors are Nourseothricin and G148. However, Ostreococcus cells are highly resistant to many antibiotics, and the concentrations of compounds necessary for selection of transgenic Ostreococcus cells is higher than for conventional model systems (2 mg/ml).
Although the process appears inefficient, the large number of cells and plasmid DNA needed for this procedure poses no significant obstacle. A larger limitation is that every generated line that is resistant to the selectable marker needs to be analysed individually to check that the entire construct is integrated in the DNA. We have observed examples in which successful expression of the selectable marker did not correspond to insertion of the actual gene of interest; i.e. partial integrations can occur. Evidently, random insertion into the genome also means that there is no control of the insertion site using this method, and methods based on homologous recombination are currently being developed. However, the method described here is, to our best knowledge, currently the only characterised method to generate genetically modified Ostreococcus cells.
As Ostreococcus tauri has only recently been used as an experimental model organism, most methods working with these cells is likely to evolve and be refined by the growing research community involved. This protocol is intended to help people initiate work with Ostreococcus, but by no means claims to describe all there is to know about genomic transformation of this microalga. The authors trust that readers will be able to adapt this protocol to their own needs as small differences in incubators (light levels or quality) and other parameters will exist and likely have to be optimised in every individual laboratory to obtain the healthy cultures needed for this protocol. After mastering the techniques described here, vectors can be constructed to generate luminescent, fluorescent, or other tagged fusion lines under the control of a range of promotors. This exciting novel experimental platform will be useful to study how complicated biochemical problems are solved in a eukaryote of reduced complexity, in which pharmacological approaches are highly facilitated3.

<table border="1">
 <tbody>
 <tr>
 <td colspan="4">Medium constituents</td>
 </tr>
 <tr>
 <td>Chemical</td>
 <td colspan="2" >Catalogue number</td>
 <td>Supplier</td>
 </tr>
 <tr>
 <td>Instant Ocean Marine Salt</td>
 <td colspan="3">from <a href="http://amazon.co.uk" target="_blank">amazon.co.uk</a> or <a href="http://rocketaquatics.co.uk" target="_blank">rocketaquatics.co.uk</a></td>
 </tr>
 <tr>
 <td>NaNO3</td>
 <td colspan="2">S5506-250G</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td colspan="2">A9434-500G</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Glycerol 2-phosphate disodium salt hydrate</td>
 <td colspan="2">G9422-100G</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>H2SeO3</td>
 <td colspan="2">84920-250G</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Ultra pure tris</td>
 <td colspan="2">15504-020</td>
 <td>Invitrogen</td>
 </tr>
 <tr>
 <td>Na2EDTA&bull;2H2O</td>
 <td colspan="2">BPE120-500G</td>
 <td>Fisher Scientific</td>
 </tr>
 <tr>
 <td>FeCl3&bull;6H2O</td>
 <td colspan="2">157740</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Na2MoO4&bull;2H2O</td>
 <td colspan="2">31439-100G-R</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>ZnSO4&bull;7H2O</td>
 <td colspan="2">Z0251</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>CoCl2&bull;6H2O</td>
 <td colspan="2">31277</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>MnCl2&bull;4H2O</td>
 <td colspan="2">203734-5G</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>CuSO4&bull;5H2O</td>
 <td colspan="2">C8027</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Vitamin B12 (cyanocobalamin)</td>
 <td colspan="2">V2876</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Biotin </td>
 <td colspan="2">47868</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Thiamine &bull; HCl </td>
 <td colspan="2">T4625</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td colspan="2">A9518-25G</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Neomycin</td>
 <td colspan="2">N6386</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Kanamycin</td>
 <td colspan="2">K4000</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td colspan="4">Consumables for culturing</td>
 </tr>
 <tr>
 <td colspan="2">Item</td>
 <td>Catalogue number</td>
 <td>Supplier</td>
 </tr>
 <tr>
 <td colspan="2">Tissue Culture Flask T25 Suspension Cultures Vented with Pouring Channel</td>
 <td>83.1810.502</td>
 <td>Starstedt</td>
 </tr>
 <tr>
 <td colspan="2">Tissue Culture Flask T75 Suspension Cultures Vented with Pouring Channel</td>
 <td>83.1813.502</td>
 <td>Starstedt</td>
 </tr>
 <tr>
 <td colspan="2">Tissue Culture Flask T175 Suspension Cultures Vented with Pouring Channel</td>
 <td>83.1812.502</td>
 <td>Starstedt</td>
 </tr>
 <tr>
 <td colspan="2">TPP Bottle top filters, 1L, complete</td>
 <td>99950T</td>
 <td>Helena Bioscience</td>
 </tr>
 <tr>
 <td colspan="2">TPP Bottle top filters, 500ml,</td>
 <td>99500T</td>
 <td>Helena Bioscience</td>
 </tr>
 <tr>
 <td colspan="2">TPP Bottle top filters, 250ml,</td>
 <td>99250T</td>
 <td>Helena Bioscience</td>
 </tr>
 <tr>
 <td colspan="2">TPP Bottle top filters, 150ml,</td>
 <td>99150T</td>
 <td>Helena Bioscience</td>
 </tr>
 <tr>
 <td colspan="2">Salinity Meter for Marine Aquarium</td>
 <td>DSG-10</td>
 <td>Daeyoon</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="3" >Chemicals for transformation</td>
 </tr>
 <tr>
 <td>Chemical</td>
 <td>Catalogue number</td>
 <td>Supplier</td>
 </tr>
 <tr>
 <td>D-Sorbitol</td>
 <td>S6021-1KG</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>Pluronic F-68 solution</td>
 <td>P5556-100ML</td>
 <td>Sigma</td>
 </tr>
 <tr>
 <td>ClonNat</td>
 <td>51000</td>
 <td>Werner</td>
 </tr>
 <tr>
 <td>Low melting point agarose</td>
 <td>16520-050</td>
 <td>Invitrogen</td>
 </tr>
 <tr>
 <td colspan="3">Consumables for transformation</td>
 </tr>
 <tr>
 <td>Item</td>
 <td>Catalogue number</td>
 <td>Supplier</td>
 </tr>
 <tr>
 <td>Tissue Culture Flask T25 Suspension Cultures Vented with Pouring Channel</td>
 <td>83.1810.502</td>
 <td>Starstedt</td>
 </tr>
 <tr>
 <td>Tissue Culture Flask T75 Suspension Cultures Vented with Pouring Channel</td>
 <td>83.1813.502</td>
 <td>Starstedt</td>
 </tr>
 <tr>
 <td>Tissue Culture Flask T175 Suspension Cultures Vented with Pouring Channel</td>
 <td>83.1812.502</td>
 <td>Starstedt</td>
 </tr>
 <tr>
 <td> Greiner centrifuge tubes 50ml</td>
 <td>227261</td>
 <td>GBO</td>
 </tr>
 <tr>
 <td>Gene Pulser/MicroPulser Cuvettes, 0.2 cm gap, 50</td>
 <td>165-2086</td>
 <td>Bio-Rad</td>
 </tr>
 <tr>
 <td>Petri dish round PS 3 VENTS H14.2 &Phi;55 </td>
 <td>391-0895</td>
 <td>VWR International</td>
 </tr>
 <tr>
 <td>Petri dish square with four vents polystyrene sterile by irradiation clear 120mm</td>
 <td>FB51788</td>
 <td>Fisher Scientific </td>
 </tr>
 <tr>
 <td>Moonlight Blue light filter</td>
 <td>183</td>
 <td>Lee Lighting</td>
 </tr>
 </tbody>
</table>

genomic transformation of the picoeukaryote ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Watch this Scientific Journal Video about Genomic Transformation of the Picoeukaryote Ostreococcus tauri at JoVE.com

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

genomic-transformation-of-the-picoeukaryote-ostreococcus-tauri

Research

JoVE Journal

Biology

Isolation of Genomic DNA from Mouse Tails

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...