This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.

This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Georgia.
1. Preparations
<ol>
	<li>For oocyte culture, purchase or freshly prepare Minimal Essential Medium (MEM) and supplement with 3 mg/ml bovine serum albumin (BSA) as outlined in Table 1. Place a polystyrene bottle on a loading balance (tare to zero). Add all reagents, except BSA, in order and bring up the final volume with MQ-water by weight to a total of 250 g. Then add the BSA, allow to dissolve and filter sterilize. 
	Note: The described MEM medium requires equilibration and cell incubation with a medical gas mixture of 5% CO2, 5% O2 and 90% N2. However, other media (e.g., CZB) can be used that allow for cell incubation in 5% CO2 under atmospheric conditions.</li>
	<li>For oocyte microinjection, purchase or prepare M2 medium.</li>
	<li>Prepare pregnant mare&#8217;s serum gonadotropin (PMSG) to a concentration of 5 IU/100 &#181;l.</li>
	<li>Purchase and reconstitute siRNA stocks to a working concentration of 1 &#181;M.</li>
</ol>
2. Mouse Oocyte Collection
<ol>
	<li>DAY 1:&#160;To stimulate ovarian follicle development and increase the number of pre-ovulatory follicles, administer 5 IU of PMSG intraperitoneally to female mice 48 hr prior to oocyte collection.</li>
	<li>DAY 3:&#160;For oocyte collection and culture, set up 35 mm culture dishes with 3 ml of MEM/BSA supplemented with 1 &#181;g/ml milrinone. This phosphodiesterase inhibitor maintains the oocytes in prophase-I arrest and prevents germinal vesicle breakdown (GVBD). Equilibrate the culture dishes in a medical gas mixture for at least 15 min. 
	Note: Milrinone supplemented media is required throughout oocyte collection, as well as oocyte microinjection and the 24 hr culture hold period post siRNA.</li>
	<li>Retrieve ovaries from the female mice using established laboratory practices15 and transfer to a new culture dish with pre-warmed and equilibrated MEM/BSA/milrinone.</li>
	<li>Place the culture dish on the stage of a stereomicroscope for oocyte collection. Release cumulus-oocyte-complexes (COC&#8217;s) by manually puncturing the antral follicles with 27 G needles. Fix one ovary to the bottom of the culture dish, with a 27 G needle attached to a 1 ml syringe, and use a second needle to puncture all large follicles. Repeat for each ovary.</li>
	<li>Using standard mouth pipetting procedures or other means of oocyte aspiration, collect all oocytes that are surrounded by 2 or more layers of compact cumulus cells. Transfer the COC&#8217;s to a new dish and incubate at 37 &#176;C for 1 hr.</li>
	<li>Carefully remove the surrounding cumulus cells by repeated gentle pipetting with a 1 ml pipette set to an aspiration volume of 750 &#181;l. Repeat the pipetting step about 12 times and allow the oocytes to recover for 5 min. Continue in this manner until all or most of the cumulus cells have been removed. Transfer the denuded oocytes to another culture dish and allow to equilibrate at 37 &#176;C for 15 min.</li>
	<li>Allocate the denuded oocytes into three experimental groups to be used for: (i) microinjection of specific siRNAs against the target of interest, (ii) microinjection of unspecific (control) siRNAs, and (iii) a non-injected control group to account for culture conditions.</li>
</ol>
3. Oocyte Microinjection
<ol>
	<li>Set up culture dishes with 3 ml M2 media supplemented with milrinone (1 &#181;g/ml) for oocyte microinjection. Prepare culture dishes containing MEM/BSA/milrinone for washing and subsequent culture of injected oocytes. 
	Note: M2 medium contains HEPES buffer and requires pre-warming at 37 &#176;C for 15 min under atmospheric conditions. MEM requires pre-warming and equilibration using medical gas as described above.</li>
	<li>Prepare the microinjection system. Load the injection needle with 5 &#181;l of 1 &#181;M specific siRNA solution. Secure the holding pipette and injection needle to micromanipulators of the microinjection system and set up the Injection Unit with calibrated volume and pressure.</li>
	<li>Place a 200 &#181;l micro-drop of M2 media on the inside of the lid of a 3 cm culture dish and add an oocyte for set up and alignment of the microinjection system.</li>
	<li>Using the oocyte as a guide, adjust the positions of the holding and injection needles. Apply negative pressure to the holding pipette to gently secure the oocyte and adjust the position of the injection needle to the widest diameter of the cell. Adjust settings to allow microinjection of approximately 10 pl of the siRNA solution anywhere into the oocyte cytoplasm. 
	&#160;<img alt="Figure 1" src="/files/ftp_upload/53586/53586fig1.jpg" /> 
	Figure 1. Oocyte microinjection set up. (A) Representative image of a 200 &#181;l micro-drop of M2 media placed on the lid from a 3 cm culture dish, with the holding pipette and injection needle positioned on the left and right, respectively. (B) Group of prophase-I arrested oocytes (GV-stage) prior to microinjection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53586/53586fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
	&#160;</li>
	<li>Return the lid to the stereomicroscope stage and add a 200 &#181;l micro-drop of fresh M2 media. Add a group of oocytes (~10) to the micro-drop by mouth pipetting, or alternative methods, then return the lid with micro-drop to the microinjection stage.
	<ol>
		<li>Proceed to microinject the individual oocytes. Secure one oocyte with the holding pipette, slowly insert the tip of the injection needle into the cytoplasm and expel the injection volume of siRNA solution. Carefully retract the injection needle and move the injected oocyte to a position at the bottom of the micro-drop. Move unsuccessfully injected oocytes to a position at the top of the micro-drop.</li>
		<li>Repeat the microinjection procedure with each oocyte. To maintain optimal oocyte viability, microinject only a small number of oocytes at a time. Ensure that this process does not take more than a few minutes to prevent temperature and pH changes in the micro-drop.</li>
	</ol>
	</li>
	<li>Aspirate successfully injected oocytes or carefully move the lid with the micro-drop of injected oocytes to the stereomicroscope. Transfer the injected viable oocytes by mouth pipetting or alternative methods, to MEM/BSA/milrinone medium to wash and equilibrate at 37 &#176;C.</li>
	<li>Using another group of oocytes, repeat the microinjection process with a new injection needle loaded with non-specific siRNAs for the control group. Wash the oocytes in MEM/BSA/ milrinone and transfer to a separate culture dish.</li>
	<li>Culture all groups of oocytes for 24 hr in MEM/BSA/milrinone at 37 &#176;C under an atmosphere of medical gas. 
	Note: The &#8216;milrinone block&#8217; culture period is needed for efficient target mRNA/protein depletion.</li>
</ol>
4. Oocyte Culture for Meiotic Maturation
<ol>
	<li>DAY 4:&#160;For each experimental group of oocytes set-up 4 culture dishes (35 mm) with 3 ml MEM/BSA. Supplement one media dish per group with 10% (high-quality) fetal bovine serum (FBS) to use for oocyte maturation. Allow all culture dishes to equilibrate at 37 &#176;C with medical gas mixture.</li>
	<li>To release oocytes from &#8216;milrinone block&#8217;, sequentially wash the oocytes three times in dishes containing MEM/BSA and then transfer the oocytes to the maturation dish containing media with 10% FBS. Culture all oocyte groups for 17 hr at 37 &#176;C, to enable resumption and progression of meiosis.</li>
	<li>DAY 5:&#160;Prepare solutions for oocyte fixation, including: (i) 4% paraformaldehyde (PFA) in PEM buffer (100 mM PIPES [pH 6.9], 1 mM MgCl2, 1 mM EGTA) with 0.5% Triton-X 100, and (ii) PBS supplemented with 5% FBS that will be used to wash and block the oocytes. 
	Note: These solutions require pre-warming to 37 &#176;C prior to oocyte fixation.</li>
	<li>For oocyte fixation (after the 17 hr culture), swiftly transfer each experimental group of oocytes by mouth pipetting or alternative methods into individual wells (of a 4-well dish) containing 750 &#181;l of pre-warmed 4% PFA solution and incubate at 37 &#176;C for 1 hr. Subsequently wash each group of oocytes 3 times (15 min each) in 750 &#181;l of pre-warmed PBS containing 5% FBS.</li>
	<li>Block oocytes in 200 &#181;l PBS/5% FBS O/N at 4 &#176;C to minimize unspecific antibody binding.</li>
</ol>
5. Immunofluorescence Analysis
<ol>
	<li>DAY 6:&#160; 
	Note: Immunostaining is done in a multi-well plate and the oocytes are serially transferred to sequential wells, containing respective antibody and wash solutions. Either 48 or 96-well plates can be used, simply adjust the solution volume based on well size. For 96-well plates use 200 &#181;l per well. Oocytes are transferred to the different solutions in the following order: primary antibody solution - 3 wash wells - secondary antibody solution - 3 wash wells. To limit light exposure the plate can be covered with foil. 
	Prepare fresh PBS/5% FBS and use this solution to prepare antibody dilutions (e.g., rabbit anti-pericentrin (1/1,000), mouse anti-tubulin (1/1,000)).</li>
	<li>Transfer the fixed oocytes of each experimental group to individual wells containing 200 &#181;l (or 100 &#181;l, if less volume is desired) of the primary antibody solution and cover the well with parafilm. Incubate according to optimal antibody-specific conditions (e.g., 37 &#176;C for 1 hr, or 4 &#176;C O/N). 
	Note: Primary antibody dilution as well as specific incubation time and temperature require testing to determine the optimal conditions for different antibodies used.</li>
	<li>After incubation with the primary antibody, wash the oocytes three times in PBS/5% FBS (10-15 min each).</li>
	<li>Transfer the oocytes into the solution containing fluorescently-conjugated secondary antibodies (e.g., 1/1,000 dilution of anti-rabbit and anti-mouse antibodies conjugated with fluorochromes of different wavelengths). Incubate for 1 hr at 37 &#176;C.</li>
	<li>Wash the oocytes three times in PBS/5% FBS (10 &#8211; 15 min each).</li>
	<li>After the final wash step, transfer the oocytes onto a clean glass slide and gently aspirate any excess wash solution. This will immobilize the oocytes on the glass surface. Add 8 &#181;l of mounting media (containing DAPI) and carefully overlay the mounting media with a 22 mm x 22 mm cover slip. Lower the coverslip slowly to avoid trapping air bubbles and/or damaging the oocytes. 
	Note: Alternatively, to maintain the 3-dimentional properties of the oocyte for confocal microscopy analysis, add a small volume of 100 &#181;m glass beads (mixed with petroleum jelly) to the corners of the cover slip prior to mounting on the slide.</li>
	<li>Store slides at 4 &#176;C, or proceed to the assessment of meiotic progression as well as the analysis of expression levels and subcellular protein localization using a fluorescence microscope equipped with the necessary filters to match the secondary antibodies used.</li>
</ol>

<ol>
	<li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nat Rev Genet. 13 (7), 493-504 (2012).</li><li>Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Szollosi, D., Calarco, P., Donahue, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+Centrioles+in+the+First+and+Second+Meiotic+Spindles+of+Mouse+Oocytes.">Absence of Centrioles in the First and Second Meiotic Spindles of Mouse Oocytes.</a> J Cell Sci. 11 (2), 521-541 (1972).</li><li>Manandhar, G., Schatten, H., Sutovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+Reduction+During+Gametogenesis+and+Its+Significance.">Centrosome Reduction During Gametogenesis and Its Significance.</a> Biol Reprod. 72 (1), 2-13 (2005).</li><li>Zimmerman, W. C., Sillibourne, J., Rosa, J., Doxsey, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis-specific+anchoring+of+gamma+tubulin+complexes+by+pericentrin+controls+spindle+organization+and+mitotic+entry.">Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.</a> Mol Biol Cell. 15 (8), 3642-3647 (2004).</li><li>Ma, W., Baumann, C., Viveiros, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEDD1+is+crucial+for+meiotic+spindle+stabilty+and+accurate+chromosome+segregation+in+mammalian+oocytes.">NEDD1 is crucial for meiotic spindle stabilty and accurate chromosome segregation in mammalian oocytes.</a> Dev Biol. 339 (439-450), (2010).</li><li>Ma, W., Viveiros, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+pericentrin+in+mouse+oocytes+disrupts+microtubule+organizing+center+function+and+meiotic+spindle+organization.">Depletion of pericentrin in mouse oocytes disrupts microtubule organizing center function and meiotic spindle organization.</a> Mol Reprod Dev. 81 (11), 1019-1029 (2014).</li><li>Bouniol-Baly, C., 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=Differential+Transcriptional+Activity+Associated+with+Chromatin+Configuration+in+Fully+Grown+Mouse+Germinal+Vesicle+Oocytes.">Differential Transcriptional Activity Associated with Chromatin Configuration in Fully Grown Mouse Germinal Vesicle Oocytes.</a> Biol Reprod. 60 (3), 580-587 (1999).</li><li>De La Fuente, R., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Activity+of+the+Mouse+Oocyte+Genome:+Companion+Granulosa+Cells+Modulate+Transcription+and+Chromatin+Remodeling.">Transcriptional Activity of the Mouse Oocyte Genome: Companion Granulosa Cells Modulate Transcription and Chromatin Remodeling.</a> Dev Biol. 229 (1), 224-236 (2001).</li><li>Hodgman, R., Tay, J., Mendez, R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPEB+phosphorylation+and+cytoplasmic+polyadenylation+are+catalyzed+by+the+kinase+IAK1/Eg2+in+maturing+mouse+oocytes.">CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes.</a> Development. 128 (14), 2815-2822 (2001).</li><li>De La Fuente, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+in+the+germinal+vesicle+(GV)+of+mammalian+oocytes.">Chromatin modifications in the germinal vesicle (GV) of mammalian oocytes.</a> Dev Biol. 292 (1), 1-12 (2006).</li><li>Wianny, F., Zernicka-Goetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+with+gene+function+by+double-stranded+RNA+in+early+mouse+development.">Specific interference with gene function by double-stranded RNA in early mouse development.</a> Nat Cell Biol. , 270-275 (2000).</li><li>Svoboda, P., Stein, P., Hayashi, H., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+reduction+of+dormant+maternal+mRNAs+in+mouse+oocytes+by+RNA+interference.">Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference.</a> Development. 127 (19), 4147-4156 (2000).</li><li>Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renaissance+of+mammalian+endogenous+RNAi.">Renaissance of mammalian endogenous RNAi.</a> FEBS Letters. 588 (15), 2550-2556 (1016).</li><li>Behringer, R., Gertsenstein, M., Nagy, K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2014).</li></ol>

The authors have nothing to disclose

While there are multiple methods for exogenous nucleic acid transfer into somatic cells, such as electroporation and transfection, microinjection is the optimal method for delivery of RNA molecules into transcriptionally quiescent mouse oocytes. The current protocol provides an effective approach for in vitro depletion of specific mRNAs that enable the functional testing of different spindle and/or MTOC-associated factors in oocytes. This approach results in efficient transcript depletion and is highly adaptable. Though siRNAs were used to specifically target Pcnt transcripts, similar conditions can be employed to target almost any gene of interest.
Several aspects of this protocol, including oocyte collection, culture and manipulation require practice and experience to preserve oocyte quality and to avoid technical artifacts. Fluctuations in the pH and/or temperature of the culture media must be avoided by working expediently and carefully. Microinjection of small groups of oocytes, and the availability of a heated stage in the microinjection system will limit detrimental temperature changes. The use of pre-validated siRNAs is recommended and efficient target protein depletion requires confirmation by immunofluorescence or Western blotting. Some steps will require modification to target different genes of interest. For example, the recommended 24 hr &#8216;hold period&#8217; in milrinone-supplemented medium is effective for various siRNAs. However, this time period may require adjustments for highly abundant mRNAs and/or target proteins with particularly long half-lives, or could be shortened for less abundant targets. Yet, it is important to consider that oocyte quality and meiotic potential are best preserved using hold periods of 24 hr or less. Caution with oocyte handling is also needed during fixation and immunofluorescence analysis. Oocyte fixation at 37 &#176;C limits microtubule depolymerization, which occurs with lower temperatures, and better preserves meiotic spindle structure. However, these conditions may need to be adjusted depending on the target protein of interest. In addition, optimal conditions for immunofluorescence (i.e., antibody concentrations as well as incubation time and temperature) also need to be established for all antibodies of interest, and tested rigorously prior to conducting the siRNA microinjection.
This technique may not effectively deplete highly abundant proteins within a reasonable timeframe that maintains oocyte quality and competence to undergo meiotic division, as oocyte viability can only be sustained for a limited time in culture. Hypomorph phenotypes may be observed with partial knockdown of target transcripts. However, co-injection of specific morpholinos may be used as a parallel strategy for additional inhibition of translation. There are also some limitations as to potential downstream analysis that are feasible with microinjected oocytes. For example, the required handling and extended culture can limit the oocyte capacity to undergo successful in vitro fertilization (IVF) and preclude the study of functional effects on pre-implantation embryo development. Moreover, large sample sizes are difficult to achieve with this approach that are needed for some downstream applications, such as immunoblotting or biochemical assays.
Despite some limitations, this approach promotes effective in vitro depletion of specific mRNAs that enables the functional testing of different factors in oocytes and has been used successfully by different research groups. Loss-of-function analysis in oocytes usually involves the generation of (conditional) knockout or transgenic RNAi mouse models. Both approaches are labor and time-intensive. In contrast, in vitro siRNA microinjection can be readily used for functional testing with significantly less time and resources. It is a particularly valuable approach to gain insight of a target protein&#8217;s functional implications before embarking on the task of generating a knockout/transgenic mouse model.
The combination of siRNA microinjection and immunofluorescence analysis provides a powerful analytical tool for the study of protein expression and subcellular distribution patterns during different stages of meiotic progression in response to siRNA-mediated target protein depletion. Potential differences in siRNA efficacy from oocyte to oocyte are readily identifiable and allow for the accurate correlation between successful target protein depletion and phenotypical/functional analysis. Further development of protocols for the co-injection of specific siRNA molecules with capped messenger RNAs encoding histone H2B-GFP fusion-proteins (or other fluorescently labeled markers) will enable the assessment of target protein function in real-time by live cell imaging.
In summary, with optimized conditions, siRNA microinjection for transcript silencing provides a valuable mechanistic approach to test the function of specific target proteins in mouse oocytes on a cell per cell basis, especially when combined with other powerful analytical tools such as immunofluorescence analysis. This combined approach was used to successfully deplete MTOC-associated proteins and evaluate meiotic spindle formation.

Meiosis is a unique division process that occurs in gametes (oocytes and sperm) and involves two successive divisions without intervening DNA synthesis to segregate homologous chromosomes and sister chromatids during meiosis-I and meiosis-II, respectively1. Errors in chromosome segregation during meiotic division in oocytes can result in aneuploidy, which is inherited by the embryo during fertilization. Notably, the incidence of aneuploidy in developing embryos increases with advancing maternal age and is a major cause of congenital birth defects as well as pregnancy loss in women1,2, thus, underscoring an important need to understand the molecular basis of aneuploidy during meiotic division.
During cell division, chromosome segregation is crucially dependent on assembly of the microtubule spindle apparatus and establishment of stable chromosome-microtubule interactions for correct attachment to opposite spindle poles. Importantly, meiotic spindle formation in mammalian oocytes differs from mitosis in somatic cells, and is regulated by unique microtubule-organizing centers (MTOCs) that lack centrioles3,4. Essential proteins necessary for microtubule nucleation and organization localize to oocyte MTOCs, including &#947;-tubulin that catalyzes microtubule assembly. In addition, pericentrin functions as an essential scaffolding protein, which binds and anchors &#947;-tubulin as well as other factors at MTOCs5. Notably, our studies demonstrate that depletion of key MTOC-associated proteins disrupts meiotic spindle organization and leads to chromosome segregation errors in oocytes, which are not fully resolved by the spindle assembly checkpoint (SAC)6,7. Therefore, defects in spindle stability, that do not trigger meiotic arrest, pose a significant risk in contributing to aneuploidy. Despite their essential role in spindle assembly and organization, oocyte MTOC protein composition and function remains poorly understood.
Testing the function of specific target proteins in mammalian oocytes is challenging, as the cells become transcriptionally quiescent shortly before the resumption of meiosis8,9. Hence, pre-ovulatory oocytes rely on maternal mRNA stores to resume meiosis and support meiotic division as well as the first cleavage divisions after fertilization10,11. The efficacy of RNA interference (RNAi) mediated degradation of mRNA transcripts in mammalian oocytes is well established and maternal RNAs recruited for translation during meiotic maturation are particularly amenable to siRNA targeting 12-14. Therefore, microinjection of short interfering RNAs (siRNAs) into oocytes provides a valuable approach to deplete target mRNAs for functional testing.
Here, we describe methods for the isolation of mouse oocytes and siRNA-mediated depletion of specific transcripts to test the function of an essential MTOC-associated protein, pericentrin. In addition, we describe immunofluorescence analysis conditions to evaluate meiotic spindle formation in oocytes.

<table border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant Mare&#39;s Serum Gonadotropin (PMSG)</td>
			<td>EMD Biosciences</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium (MEM)</td>
			<td></td>
			<td></td>
			<td>*Recipe outlined in Table 1</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (10x)</td>
			<td>Sigma</td>
			<td>E-7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic Acid, sodium salt&#160;</td>
			<td>Sigma</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G, potassium salt&#160;</td>
			<td>Sigma</td>
			<td>P-7794</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Sigma</td>
			<td>S-9137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Sigma</td>
			<td>G-8540</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, disodium salt dihydrate&#160;</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential Amino Acids (50x)</td>
			<td>Gibco&#160;</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamin Mixture (100x)</td>
			<td>Sigma</td>
			<td>M-6895</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red solution</td>
			<td>Sigma</td>
			<td>P-0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma</td>
			<td>M4659</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax M2 Media with Hepes</td>
			<td>EMD Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNAs targeting Pericentrin</td>
			<td>Qiagen</td>
			<td>GS18541</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control siRNAs&#160;</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pericentrin (rabbit)</td>
			<td>Covance</td>
			<td>PRB-432C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin (mouse)</td>
			<td>Sigma</td>
			<td>T-6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21430</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti -mouse Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Major Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope (SMZ 800)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Fluorescent Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Micro-injections System</td>
			<td>Eppenforf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulators</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-injection needles (femtotips)</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td></td>
		</tr>
		<tr>
			<td>Holding pipettes (VacuTip)</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm culture dishes</td>
			<td>Corning Life Sciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3367</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm CA Filter System</td>
			<td>Corning Life Sciences</td>
			<td>430768</td>
			<td></td>
		</tr>
	</tbody>
</table>

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Microinjection of siRNAs provides an effective approach for mRNA degradation and subsequent protein depletion in oocytes, which enables efficient and highly specific functional testing of different target factors in vitro. Subsequently, immunofluorescence is used for specific phenotype analysis as well as to validate protein depletion in siRNA-injected oocytes. In the current example, fluorescent labeling of individual oocytes with DAPI together with anti-tubulin and anti-pericentrin antibodies enabled: (i) confirmation of pericentrin depletion as well as (ii) a comprehensive assessment of both chromatin and meiotic spindle configurations in control and PCNT-depleted oocytes.
Representative immunofluorescence images of oocytes microinjected with non-specific control or Pcnt siRNAs are shown in Figure 27. Following a 17 hr culture, most control oocytes reached the metaphase-II stage and contain organized meiotic spindles with aligned chromosomes and bright pericentrin labeling at the spindle poles (Figure 2A). Notably, pericentrin was not detected in oocytes injected with Pcnt siRNA, confirming efficient protein knockdown in these cells. Moreover, the majority of PCNT-depleted oocytes remained at the metaphase-I stage and exhibit disorganized spindle structures with misaligned chromosomes (Figure 2B). The few PCNT-depleted oocytes that progressed to metaphase-II also exhibited disrupted spindles (Figure 2C).
<img alt="Figure 2" src="/files/ftp_upload/53586/53586fig2.jpg" /> 
Figure 2. Immunofluorescence analysis of meiotic spindle organization in mouse oocytes. Representative images of oocytes injected with either (A) control non-specific siRNAs or with (B, C) specific Pcnt-siRNAs. Oocytes were double-labeled with anti-pericentrin (red) together with an anti-acetylated &#945;-tubulin antibody for the detection of microtubules (green). DNA was labeled with DAPI and is shown in blue. The inset shows a 2X magnification of the spindle pole area. Arrows denote misaligned chromosomes. *Spindle pole. Pb: First polar body. Scale bar of 10 &#181;m. This figure has been modified from Ma and Viveiros, 20147. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53586/53586fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Recipe for Preparation of MEM. Listing of all reagents and specific amounts needed to prepare 250 ml of MEM. The media is filter sterilized using a 0.45 &#956;m cellulose acetate (CA) membrane filter unit and stored at 4 &#176;C for a maximum of 7 days.
<table border="0">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Amount&#160;</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (10x)</td>
			<td>25 ml</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>0.550 g</td>
		</tr>
		<tr>
			<td>Pyruvic Acid&#160;</td>
			<td>0.0063 g</td>
		</tr>
		<tr>
			<td>Penicillin G&#160;</td>
			<td>0.0188 g</td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>0.0125 g</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>0.073 g</td>
		</tr>
		<tr>
			<td>EDTA, Disodium Salt Dihydrate&#160;</td>
			<td>0.1 mg</td>
		</tr>
		<tr>
			<td>Essential Amino Acids (50x)</td>
			<td>5.0 ml</td>
		</tr>
		<tr>
			<td>MEM Vitamin Mixture (100x)</td>
			<td>2.5 ml</td>
		</tr>
		<tr>
			<td>Phenol Red Solution</td>
			<td>0.2 ml</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)&#160;</td>
			<td>0.75 g</td>
		</tr>
	</tbody>
</table>

Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

meiotic-spindle-assessment-mouse-oocytes-sirna-mediated

Nanyang Technological University

Research

JoVE Journal

Biology

11.6K Views.  College of Veterinary Medicine, University of Georgia. Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Video: Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Summary

Explore More Videos

Chapters in this video

Nuclear Transfer into Mouse Oocytes

Retrieval of Mouse Oocytes

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Visualizing RNA Localization in Xenopus Oocytes

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Flow Cytometry Purification of Mouse Meiotic Cells

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca²⁺ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy