Name: mouse oocyte microinjection, maturation and ploidy assessment
Uploaded: 2011-07-23T14:00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about Mouse Oocyte Microinjection, Maturation and Ploidy Assessment at JoVE.com

This work was conducted in Richard M. Schultz's laboratory. The authors would also like to acknowledge Michael Lampson for conceptualizing the centromere-counting assay and access to his confocal microscope. Teresa Chiang and Francesca Duncan assisted in optimizing the centromere-counting assay. Paula Stein is supported by HD022681 (to RMS) and Karen Schindler is supported by HD055822.

1. Mouse oocyte collection
<ol>
<li>To maximize the number of antral follicles isolated from each mouse, intraperitoneally inject sexually mature female mice (we use 6-week-old CF-1 mice from Harlan) with 5 IU of pregnant mare's serum gonadotropin (PMSG).</li>
<li>Prepare the collection medium (MEM/PVP) (3 ml/mouse) by adding milrinone to 2.5 &mu;M and warm it at 37&deg;C. Milrinone is a phosphodiesterase inhibitor that maintains meiotic arrest once oocytes are removed from follicles. Alternatively, 3-isobutyl-1-methylxanthine (IBMX) (0.2 mM) or dibutyryl-cyclic adenosine monophosphate (dbcAMP) (100 &mu;g/ml) can be used. Prepare the culture medium by adding glutamine (1 mM) and milrinone (2.5 &mu;M) to 1 ml of CZB and allow it to equilibrate in the incubator for at least an hour. Set up the microdrops of each in a Petri dish and overlay with mineral oil. The CZB dish is placed into the incubator while the MEM/PVP dish remains outside on a slide warmer. Other collection and culture medium that are commercially available (M2 and M16 from Specialty media or Sigma) are also routinely used.</li> 
<li>Approximately 48 h after PMSG priming, sacrifice the mouse using CO2 sedation followed by cervical dislocation, dissect the ovaries and place them into a watchglass containing pre-warmed MEM/PVP + milrinone (MEM/PVP+M).</li>
<li>Using a 1 ml insulin syringe, anchor the ovaries to the dish and release the antral follicles by puncturing them several times with 27-gauge (or sewing) needles that are fastened together.</li> 
<li>While looking through a dissection microscope, collect cumulus-oocyte complexes using a mouth-operated glass pipette. It is helpful to have a microscope with 
a lot of contrast. Alternatively, the medium containing the tissue and cells can be pipetted on to a lid of a plastic Petri dish. Only collect large, antral 
follicles and not smaller pre-antral follicles or denuded oocytes. Once collected, transfer the complexes to a microdrop of MEM/PVP+M that is on the slide warmer 
and under oil.</li>
<li>Using a small pipette (slightly larger than the diameter of the oocyte), pipette up and down the complexes to detach the cumulus cells. Transfer the denuded oocytes with a larger pipette into a microdrop of CZB + milrinone (CZB+M) and place in the incubator.</li>
<li>Allow oocytes to recover for at least 1 h in the incubator prior to manipulation.</li>
</ol>
2. Oocyte microinjection
<ol>
<li>Make injection pipettes by pulling borosilicate-glass capillary tubing in a mechanical puller. We use a Flaming-Brown micropipette puller (Model P-97) with the following settings: P=500, Heat=300, Pull=150, Vel=100, Time=150.</li>
<li>Prepare the microinjection platform by placing a 5 &mu;l drop of MEM/PVP+M as close as possible to a 0.5 &mu;l drop of injection material of choice (e.g. siRNA, cRNA, morpholino oligonucleotide, etc.) on a 1-well chamber slide that has the media chamber removed. Cover with mineral oil and place on the microscope stage (Fig. 1).</li>
<li>Place injection and holding pipettes into the holders and position into the drop of MEM/PVP+M. Turn on the nitrogen tanks that are connected to the picoinjector and anti-vibration table. Open the tip of the injection pipette by gently tapping it against the holding pipette while filling it with medium. If the opening is too large, the medium will move in and out quickly and the needle will kill the cell. Pipettes with too small of an opening often clog easily.</li>
<li>Transfer oocytes (5-10) from the incubator to the MEM/PVP+M drop on the platform. Before injecting, setup the picoinjector. We use the following parameters on our picoinjector (Model PLI-100 from Harvard Apparatus): PBal=2.5-3.5 psi, PInj=7.8 psi, PClear= 10-12 psi, time= 3s. This results in an injection volume of 5-10 pL.</li> 
<li>While pressing CLEAR, move the injection needle to the material drop and fill. Return it to the medium drop.</li>
<li>Use the holding pipette to capture an oocyte and align the injection needle, oocyte and holding pipette along the x-axis.</li> 
<li>Under higher magnification, advance the injection pipette through the oocyte being careful to avoid the nucleus. Once the plasma membrane is pierced, press INJECT. After injection, withdraw the needle. Release the oocyte and repeat. Once all the oocytes are injected, return them to the incubator. Hold in CZB+M for desired amount of time; this time is dependant upon the experimental aim (i.e. siRNA and morpholino knockdown (up to 24 h), overexpression (3-12 h)). Please note that incubations longer than 24 h will compromise meiotic maturation.</li>
</ol>
3. Oocyte maturation
<ol>
<li>After incubation wash the oocytes through several drops of CZB (maturation medium) and transfer to a microdrop of maturation medium under oil and place in the incubator.</li> 
<li>Allow 16 h for complete maturation to metaphase of meiosis II.</li>
</ol>
4. Ploidy analysis
<ol>
<li>Prepare CZB + monastrol (100 &mu;M) and place 750 &mu;l into the well of an organ culture dish that has water in the outer ring.</li>
<li>Wash eggs through several drops of CZB + monastrol and transfer them into the organ culture dish. Place in the incubator for 1 h.</li>
<li>Fix the eggs by transferring them into a watchglass containing 2% paraformaldehyde in PBS for 20 minutes at room temperature. Transfer into another watchglass with blocking solution. This dish can be stored at 4&deg; C until processed.</li>
<li>Transfer to permeabilization solution (PBS + 0.3% BSA + 0.1% TritonX-100 + 0.02% NaN3) in a watchglass for 15 minutes at room temperature. Rinse through several large volumes of blocking solution (PBS + 0.3% BSA + 0.01 Tween-20 + 0.02% NaN3).</li>
<li>The rest of the procedure is performed on the lid of a 96-well plate in a humidified chamber at room temperature that is protected from light. The drops (&tilde;25 &mu;l) are placed inside the circular indentations. Transfer eggs to blocking solution for 15 minutes.</li> 
<li>To label centromeres, transfer eggs to a drop of blocking solution containing CREST anti-serum at 1:40. Incubate for at least 1 h. Wash through 3 drops of blocking solution, incubating 15 minutes in each.</li>
<li>Transfer eggs to a drop of blocking solution containing Cy5- or Alexa-fluor-594-conjugated anti-human IgG (1:200) and incubate for 1h. Repeat washing steps as above except for the last step, include Sytox Green (1:5,000) in the blocking solution to detect chromosomes. Mount in 5 &mu;l of Vectashield. To prevent crushing of the eggs, put 4 small spots of petroleum jelly at the corners of where the coverslip will be. Place a coverslip on top and seal with nail polish. Store in a slide box at 4&deg; C until processing via confocal microscopy.</li>
<li>While imaging with a 100X objective, capture 0.4 &mu;m steps in the Z-plane and image the entire region of the metaphase spindle. Count centromeres using imaging software such as Image J (NIH).</li>
</ol>
5. Representative Results: 
Figure 2 is a Z-projection from a euploid egg. At metaphase of MII, euploid mouse eggs contain 20 pairs of chromosomes and therefore have 40 centromeres. Occasionally chromosomes fail to spread despite monastrol treatment. This situation makes it difficult to reliably count centromeres and we therefore do not include these eggs in our analyses. Occasionally, it may be challenging to determine whether a CREST-immunoreactive "spot" is 1 or 2 centromeres. Using programs such as Image J is useful because one can analyze each Z-section while carefully noting the orientation of the chromosomes, the number of sections in which a "spot" is detected and the pixel intensity the "spots" have. Depending where the meiotic spindle is positioned relative to the polar body, the regions of DNA can overlap and these samples should not be included in the analysis.
Unmanipulated, in vivo ovulated eggs from reproductively young mice have low rates of aneuploidy (&tilde;1-2%). However, for reasons not understood, microinjection and in vitro maturation procedures can increase this rate upwards of 10%. Therefore, it is critical that control injected oocytes are included in any microinjection study.
<img src="/files/ftp_upload/2851/2851fig1.jpg" alt="Figure 1"/> 
Figure 1. Microinjection dish set up. A chamber slide with 5 &mu;l drops of MEM/PVP + M just above 0.5&mu;l drops of injection solution. Cover with mineral oil. In this example there are 3 drops for 3 different injection solutions and the slide is sitting on the stage of a microscope. To the left is the microinjection needle and to the right is the holding pipette. Note that there is a reflection of the needle holders.
<img src="/files/ftp_upload/2851/2851fig2.jpg" alt="Figure 2"/> 
Figure 2. Ploidy results. A Z-projection of a euploid metaphase II egg. DNA is colored in green and kinetochores are colored in red. The arrows point to 2 distinct chromatid arms indicating that kinetochores (#17 and #18) overlap. Euploid mouse eggs contain 20 kinetochore pairs (40 total "spots"). An aneuploid egg would contain any variation on this number. If this procedure were conducted on metaphase MI oocytes, there would be 40 kinetochore pairs (80 total "spots").
<img src="/files/ftp_upload/2851/2851table1.jpg" alt="Table 1"/> 
Table 1. Recipe for collection and microinjection medium (MEM/PVP + M). All materials are embryo-culture grade and from Sigma-Aldrich. Filter sterilize through 0.22&mu;m PVDF filters (we use Milipore Stericups) and store at 4&deg;C.
<img src="/files/ftp_upload/2851/2851table2.jpg" alt="Table 2"/> 
Table 2. Recipe for CZB medium. All materials are embryo-culture grade and from Sigma-Aldrich. Filter sterilize through 0.22&mu;m PVDF filters (we use Milipore Stericups) and store at 4&deg;C.

<ol>
	<li>Hunt, P. A., Hassold, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 296 (5576), 2181-2181 (2002).</li><li>Hassold, T., Hall, H., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hum Mol Genet. 16 Spec No. 2, R203-R203 (2007).</li><li>Chatot, C. L., Ziomek, C. A., Bavister, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of reproduction and fertility. 86 (2), 679-679 (1989).</li><li>Schultz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oogenesis and the control of meiotic maturation. , (1986).</li><li>Moore, G. P., Lintern-Moore, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biol Reprod. 18 (5), 865-865 (1978).</li><li>Hodges, C. A., 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=.">.</a> Chromosoma. 111 (3), 165-165 (2002).</li><li>Schindler, K., 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=.">.</a> Cell Cycle. 8 (7), 1090-1090 (2009).</li><li>Chiang, T., Duncan, F. E., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 20 (17), 1522-1522 (2010).</li><li>Mayer, T. U., Kapoor, T. M., Haggarty, 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=.">.</a> Science. 286 (5441), 971-971 (1999).</li><li>Stein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor protocols. 2009 (1), pdb prot5135-pdb prot5135 (2009).</li><li>Stein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor protocols. 2009 (1), pdb prot5132-pdb prot5132 (2009).</li><li>Reis, A., Chang, H. Y., Levasseur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Cell Biol. 8 (5), 539-539 (2006).</li><li>Schuh, M., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 130 (4), 484-484 (2007).</li><li>Backs, J., Stein, P., Backs, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (1), 81-81 (2010).</li><li>Schindler, K., 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=.">.</a> Biol Reprod. 80 (4), 795-795 (2009).</li><li>Kudo, N. R., Wassmann, K., Anger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 126 (1), 135-135 (2006).</li><li>Shoji, S., Yoshida, N., Amanai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The EMBO Journal. 25 (4), 834-834 (2006).</li></ol>

No conflicts of interest declared.

Microinjection of oocytes is a powerful method to study mechanisms that regulate meiotic maturation 10,11, 12, 13. This method provides an economical way to test hypotheses prior to making a large investment in developing transgenic and targeted mouse models. Oocyte collection and microinjection techniques require more time to master than typical cell biology procedures. Specific obstacles with collection often include controlling the mouth pipette, pulling the proper size glass pipette for collection and stripping of the somatic cells and increasing collection speed to minimize the time in which oocytes are outside of the incubator. We recommend practicing many times prior to doing experiments. Transferring oocytes between microdrops while maintaining the same number of cells is a great way to become comfortable with this method.
Cell death commonly occurs while learning microinjection. This could occur for a number of reasons, including injection of too large of a volume of material (i.e. the injection needle opening is too big), hitting the nucleus with the injection needle, piercing the opposite side of the oocyte or that the material injected is toxic to the oocyte. Practicing with injecting buffer into oocytes until your survival rate is at least 50% is key to mastering this technique. If oocytes fail to mature it is likely that the milrinone was not diluted enough. We recommend rinsing the oocytes through many large drops of milrinone-free CZB before maturation.
The ploidy analysis following microinjection is one of many assays to assess meiotic maturation. Other routine analyses we use in the laboratory include monitoring the kinetics by which the oocytes progress through meiosis, immunofluorescence to analyze spindle formation and chromosome alignment and egg activation or in vitro fertilization to assess the developmental consequences of the oocyte manipulation 14,15, 16, 17.

Table of specific reagents:
<table border="1">
 <tbody>
 <tr>
 <th>Name of Reagent</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Milrinone</td>
 <td>Sigma-Aldrich</td>
 <td>M4659</td>
 <td>Resuspend in DMSO at 2.5mM</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>Sigma-Aldrich</td>
 <td>M5310</td>
 <td>Only use embryo-tested, sterile-filtered</td>
 </tr>
 <tr>
 <td>CREST autoserum</td>
 <td>Immunovision</td>
 <td>HCT-0100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sytox Green</td>
 <td>Invitrogen</td>
 <td>57020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-human Alexa 594</td>
 <td>Invitrogen</td>
 <td>A-11014</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vectashield</td>
 <td>Vector Laboratories</td>
 <td>H-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Polysciences</td>
 <td>577773</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Albumin from bovine serum</td>
 <td>Sigma-Aldrich</td>
 <td>A3294</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PMSG</td>
 <td>Calbiochem</td>
 <td>367222</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Monastrol</td>
 <td>Sigma-Aldrich</td>
 <td>M8515</td>
 <td>Resuspend in DMSO at 100mM</td>
 </tr>
 <tr>
 <td>Tween-20</td>
 <td>Sigma-Aldrich</td>
 <td>274348</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TritonX-100</td>
 <td>Sigma-Aldrich</td>
 <td>X-100</td>
 <td>&nbsp;</td>
 </tr> 
 </tbody>
</table>
Table of specific equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of Equipment</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Mouthpiece</td>
 <td>Biodiseno</td>
 <td>MP-001-Y</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Watchglass</td>
 <td>Electron Microscopy Sciences</td>
 <td>70543-30</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>B-D</td>
 <td>309623</td>
 <td>1ml, 27G1/2</td>
 </tr>
 <tr>
 <td>60 mm Petri Dish</td>
 <td>Falcon</td>
 <td>351007</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass Pasteur pipets</td>
 <td>Fisher scientific</td>
 <td>13-678-200</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Side warmer</td>
 <td>Fisher scientific</td>
 <td>&nbsp;</td>
 <td>Any standard model</td>
 </tr>
 <tr>
 <td>Dissection microscope</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Any standard model</td>
 </tr>
 <tr>
 <td>Chamber Slide</td>
 <td>Nunc</td>
 <td>177372</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Capillary Tubing</td>
 <td>Drummond</td>
 <td>1-000-0500</td>
 <td>microcaps</td>
 </tr>
 <tr>
 <td>Pipette Puller</td>
 <td>Flaming-Brown micropipette puller </td>
 <td>&nbsp;</td>
 <td> Model P-97</td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>Any standard model </td>
 </tr>
 <tr>
 <td>Micromanipulators</td>
 <td>Eppendorf</td>
 <td>&nbsp;</td>
 <td>Any standard model </td>
 </tr>
 <tr>
 <td>Picoinjector</td>
 <td>Harvard Apparatus</td>
 <td>Model PLI-100 </td>
 <td>Any standard model</td>
 </tr>
 <tr>
 <td>CO2 tanks</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For incubator</td>
 </tr>
 <tr>
 <td>N2 tank</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For table and injector</td>
 </tr>
 <tr>
 <td>Anti-vibration table</td>
 <td>Technical Manufacturing</td>
 <td>&nbsp;</td>
 <td>Any standard model </td>
 </tr>
 <tr>
 <td>Incubator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Any standard model </td>
 </tr>
 <tr>
 <td>Holding pipettes</td>
 <td>Eppendorf</td>
 <td>930001015</td>
 <td>Vacutip</td>
 </tr>
 <tr>
 <td>Confocal microscope</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>Any standard model </td>
 </tr>
 <tr>
 <td>Dissection tools</td>
 <td>Fine science tools</td>
 <td>&nbsp;</td>
 <td>Any standard model </td>
 </tr>
 <tr>
 <td>Humidified chamber</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>We use tupperware</td>
 </tr>
 <tr>
 <td>Lid of 96 well plate</td>
 <td>Nunc</td>
 <td>263339</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope slides</td>
 <td>Fisher scientific</td>
 <td>12-544-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Coverslips</td>
 <td>Thomas scientific</td>
 <td>6663-F10</td>
 <td>Thickness will vary for particular microscopes</td>
 </tr>
 <tr>
 <td>Center well organ culture dish</td>
 <td>Fisher scientific</td>
 <td>353037</td>
 <td>60 X 15mm</td>
 </tr>
 </tbody>
</table>

mouse oocyte microinjection, maturation and ploidy assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Watch this Scientific Journal Video about Mouse Oocyte Microinjection, Maturation and Ploidy Assessment at JoVE.com

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

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In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

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.

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...