This work was supported by a Research Grant from the American Society for Reproductive Medicine and from the Charles and Johanna Busch Memorial Fund at Rutgers, The State University of NJ to K.S. A.L.N. was supported by a grant from the N.I.H. (F31 HD0989597).

1. Molecular Cloning
<ol>
	<li>Obtain full-length coding sequence of the gene of interest and the plasmid in vitro transcription (pIVT) vector11. 
	NOTE: Full-length cDNA clones are commercially available from various vendors or can be generated via reverse transcription polymerase chain reaction (RT-PCR). The National Center for Biotechnology Information (NCBI) online resource provides transcript sequences for genes from the Nucleotide and Expressed sequence tagged (EST) databases. For ease of protein visualization and analysis of expression levels, fusion to green fluorescent protein (Gfp) or another suitable fluorescent tag may be desired in the cloning strategy.</li>
	<li>Using a validated cloning strategy, insert the coding sequence of the gene of interest into the multiple cloning region of pIVT. A detailed description of molecular cloning and the required steps have previously been described 12. 
	NOTE: If a gene-fusion product is to be generated, be sure that the cloning is in the proper reading frame.</li>
	<li>Sequence the construct using Globin primers to ensure correct gene insertion, proper in-frame fusion, and that no polymerase-induced point mutations were created. 
	NOTE: If Sanger DNA sequencing equipment is not available, many companies offer low cost DNA sequencing options.</li>
	<li>Mutagenize DNA if generating single nucleotide polymorphisms (SNPs) or insertions/deletions (INDELS). DNA mutagenesis is described in steps 1.5-1.7 below. For wild-type constructs, proceed with the linearization step 1.7.</li>
	<li>Design mutagenesis primers and complete the mutagenesis PCR reaction to generate mutant construct per manufacturer&#39;s protocol. PCR-mediated site-directed mutagenesis has been previously described 13. Additionally, many companies offer site-directed mutagenesis kits which include protocols.</li>
	<li>Transform mutagenized DNA into bacterial host. Isolate and purify plasmid. 
	NOTE: Many companies make kits that can be used to easily isolate and purify plasmid DNA. Follow manufacturers protocol accordingly. If using a gram-negative bacterial strain such as E. coli, it is recommended to use a kit that removes endotoxins.</li>
	<li>Sequence the isolated DNA from bacterial transformants using standard Sanger DNA sequencing to confirm introduction of the desired SNP or INDEL.</li>
	<li>Linearize final products using single restriction enzyme digestion of the purified DNA. 
	NOTE: The pIVT vector contains multiple single-enzyme cut sites listed in the plasmid map on Addgene 14.</li>
	<li>Ensure that the product is linear via agarose gel electrophoresis. The methodology for agarose gel electrophoresis has previously been defined 14. 
	NOTE: For all remaining steps, use barrier (filter) pipet tips and RNase-free materials to ensure production of stable, high quality RNA.</li>
	<li>Purify the digested product using a validated DNA clean-up protocol or kit, and elute in 30 &#181;L RNase/DNase-free water. 
	NOTE: Silica-matrix spin column-based kits are recommended for high quality, purified DNA. Follow manufacturers protocol accordingly.</li>
	<li>In vitro transcribe DNA using T7 polymerase following the manufacturer&#39;s protocol.</li>
	<li>Purify and elute RNA in 30 &#181;L of RNase/DNase free water using a silica-matrix spin column-based nucleic acid purification kit following the manufacturer&#39;s protocol.</li>
	<li>Perform denaturing agarose gel electrophoresis using formaldehyde to confirm the final RNA product size. See steps 1.13-1.22 below 14. (Figure 1).</li>
	<li>Prepare gel by heating 1 g agarose in 72 mL water until dissolved, then cool to 60 &#176;C.</li>
	<li>Prepare 10X MOPS running buffer by adding 0.4 M MOPS (pH 7.0), 0.1 M sodium acetate, and 0.01 M EDTA.</li>
	<li>Add 10 mL of 10X MOPS running buffer, and 18 mL of 37% formaldehyde (12.3 M) to the cooled agarose solution.</li>
	<li>Caste the gel by pouring the agarose solution into a gel-forming container. Use a comb large enough to accommodate 10 &#181;L. After gel has set and is firm to the touch, gently remove the comb.</li>
	<li>Assemble the gel in the electrophoresis tank, and add enough 1X MOPS running buffer, ensuring the gel is completely covered.</li>
	<li>Prepare RNA sample by adding 1 &#181;g of RNA to 0.5X volumes of formaldehyde-containing loading dye.</li>
	<li>Add ethidium bromide to RNA solution at a concentration of 10 &#181;g/mL.</li>
	<li>Denature RNA sample solution at 70 &#176;C for 5 min.</li>
	<li>Using sterile, 10 &#181;L filter tips, load 5 &#181;L of molecular weight marker and 5 &#181;L of RNA sample(s) into separate wells of the gel and electrophorese at 5 V/cm until the dye has migrated at least two-thirds of the length of the gel.</li>
	<li>Visualize the RNA in the gel on a UV trans illuminator. Ensure the RNA is the correct size by comparing to the molecular weight marker.</li>
	<li>To measure the concentration and purity of the RNA, transfer 1.2 &#181;L of purified RNA using sterile 10 &#181;L filter tips onto a UV-spectrophotometer. 
	Note that high quality RNA should have absorbance ratios of A260/280 (1.8-2.2) and 260/230 (&#62;1.7) respectively.</li>
	<li>Using 10 &#181;L filter tips, aliquot the remaining purified RNA into sterile 0.5 &#181;L centrifuge tubes (3-5 &#181;L/tube). Store tubes at -80 &#176;C until ready for microinjection. 
	NOTE: A final injection concentration of 500 ng/&#181;L is an optimal starting point. It is recommended to dilute RNA 1:2 in RNase free H20 prior to microinjection to reduce stickiness of RNA in microinjection pipette.</li>
</ol>
2. Kinetochore-Microtubule Attachment Assay
<ol>
	<li>Divide oocytes into equal groups for microinjection. 
	NOTE: Oocyte collection, transfer, microinjection, and meiotic maturation has been described in detail in JoVE previously 15.</li>
	<li>Microinject one group with experimental mRNA and the other group(s) with the WT control and PBS or Gfp mRNA. 
	NOTE: 500 ng/&#181;L is the recommended injection concentration to begin with 10. A picoinjector can be used to control injection amounts. An injection volume of 5-10 pL is recommended 15.</li>
	<li>Using a hand or mouth operated pipetting apparatus where the glass pipette opening is slightly larger than the diameter of an oocyte (100-150 &#181;m), transfer oocytes into a 100 &#181;L drop of milrinone-free culture medium in a Petri dish covered in embryo quality mineral oil and incubate oocytes for 7 h at 37 &#176;C in 5% C02 to allow sufficient maturation to metaphase of meiosis I (Met I) 15.</li>
	<li>After incubation, using the same pipette apparatus as above, transfer oocytes into a center-well organ culture dish containing 700 &#181;L of pre-chilled minimum essential media (MEM) collection medium in the center well and 500 &#181;L H20 in the outside ring and place dish on ice for 7 min. 
	NOTE: It is recommended to pre-chill MEM media and center well organ culture dishes at -20 &#176;C for at least 20 min prior to treatment of oocytes.</li>
	<li>Fix the oocytes by transferring them using the pipette apparatus into a well of a clear glass spot plate containing 400 &#181;L of 2% paraformaldehyde in 1X PBS for 20 min at room temperature.</li>
	<li>Using same pipette apparatus, transfer the oocytes into another well on a spot plate containing 400 &#181;L blocking solution (PBS + 0.3% BSA + 0.01% Tween-20 + 0.02% NaN3) and store at 4 &#176;C until ready to process for immunostaining. 
	NOTE: For storing oocytes at 4 &#176;C, cover glass spot plate with parafilm to prevent evaporation.</li>
	<li>Using same pipette apparatus, transfer oocytes to a new well on a spot plate containing 400 &#181;L permeabilization solution (PBS + 0.3% BSA + 0.1% TritonX-100 + 0.02% NaN3) and incubate at room temperature for 20 min. Transfer oocytes quickly through three drops of 400 &#181;L blocking solution to remove any residual detergent. 
	NOTE: A 9-well clear glass spot plate works well for moving groups through multiple treatments on a single dish (steps 2.4-2.5).</li>
	<li>Perform the remaining immunocytochemistry procedures in a humidified chamber, protected from light at room temperature. Use a medium-sized plastic container lined with damp paper towels and cover with aluminum foil.</li>
	<li>Using the pipette apparatus, transfer oocytes to 30 &#181;L blocking solution on the lid of a 96-well plate and place within the humidified chamber for 10 min. 
	NOTE: Drops are placed in the circular indentions on the plate.</li>
	<li>To label centromeres and spindle, using the pipette apparatus, transfer oocytes to 30 &#181;L blocking solution containing anti-centromeric antigen (ACA, Crest) (1:30) and anti-acetylated tubulin (1:1000) and incubate for at least 1 h.</li>
	<li>Rinse oocytes by transferring through three 30 &#181;L drops of blocking solution for 10 min each.</li>
	<li>Using the pipette apparatus, transfer oocytes to a 30 &#181;L drop of blocking solution containing secondary antibodies complimentary to the antibodies used above (anti-human IgG 1:200, anti-mouse IgG 1:200) and incubate for 1 h.</li>
	<li>Repeat the 3, 10-min rinsing steps in 30 &#181;L blocking solution as described in step 2.11.</li>
	<li>Transfer the oocytes, using the pipette apparatus, into 3-5 &#181;L mounting medium containing DAPI (5.9 &#181;g/&#181;L) on a glass microscope slide.</li>
	<li>Place 4 small drops (size of a pin head) of petroleum jelly on the corners of the cover slip to prevent crushing of the oocytes.</li>
	<li>Gently place the cover slip petroleum-jelly side down onto the slide.</li>
	<li>Seal coverslip edges with clear nail polish and allow to dry for 5 min.</li>
	<li>Store slides at 4 &#176;C, protected from light, until processing via confocal microscopy. 
	NOTE: Ensure the refractive index of mounting medium, and coverslip, matches the microscope objectives being utilized. Recommended mounting materials have previously been described 15.</li>
	<li>Image oocytes using a 40-63x objective on a confocal microscope, capturing small steps (&#8804; 1 &#181;m) in the z-plane, optimized for the working objective, and image the entire region of the metaphase spindle. 
	NOTE: Make sure to image all 3 channels based on specific secondary antibodies used in step 2.11. Image averaging &#8805; 4 and a zoom of 3 are ideal for adequate resolution. A 1 &#181;m step size provides clear visualization of microtubules and kinetochores in mouse oocytes. The procedure for image capture will vary from microscope to microscope. Please refer to the manufacturers confocal user guide for specific steps on how to configure microscope settings.</li>
	<li>Identify kinetochore-microtubule attachment status using imaging software following steps 2.21-2.23 below. 
	NOTE: The following steps describe this procedure using Image J (NIH) free downloadable software, however, alternate imaging software can be used. Attachment status types have previously been defined 16 .</li>
	<li>Open image by dragging file into Image J tool bar.</li>
	<li>In the opened z-series, make all channels visible (DNA, kinetochore, and spindle) by clicking &#34;merge channels&#34;, available in the &#34;Image&#34; drop-down menu, under the &#34;color&#34; sub tab.</li>
	<li>Using the toggle at the bottom of the image, move through each z-slice and determine kinetochore microtubule attachment. Detailed attachment descriptions have been previously described 16</li>
</ol>
3. Chromosome Alignment Challenge Assay
<ol>
	<li>Using the pipette apparatus as described in step 2.3, transfer oocytes into a 100 &#181;L drop of milrinone-free culture medium under oil and incubate oocytes for 7 h to allow sufficient maturation to metaphase of meiosis I (Met I) as previously described in step 2.3 17. 
	NOTE: Oocyte collection, microinjection, and maturation procedures were outlined previously 15. Refer to the attachment assay protocol in steps 2.1-2.2 for controls and for RNA microinjection concentrations.</li>
	<li>Using same pipette apparatus, transfer oocytes to a center-well organ culture dish containing 700 &#181;L culture medium containing monastrol [100 &#181;M] in the center well and 400 &#181;L H20 in the surrounding ring and incubate at 37 &#176;C for 2 h.</li>
	<li>Rinse out monastrol by transferring oocytes, using the pipette apparatus as above, through three drops of 100 &#181;L CZB culture medium, and then transfer the oocytes to a new center-well organ culture dish containing 700 &#181;L culture medium + MG132 [5 &#181;M] for 3 h in the center ring. Add 400 &#181;L H20 to the outside ring.</li>
	<li>Fix the oocytes by transferring them, using the pipette apparatus, into a well of a clear glass spot plate containing 400 &#181;L 2% paraformaldehyde in PBS for 20 min at room temperature.</li>
	<li>After fixation, transfer the oocytes, using the pipette apparatus, into a new well of a clear glass spot plate containing 400 &#181;L blocking solution and store at 4 &#176;C until processed for immunostaining. 
	NOTE: For storing oocytes at 4 &#176;C, cover glass spot plate with parafilm to prevent evaporation.</li>
	<li>Permeabilize and label oocytes via immunocytochemistry as described in steps 2.6-2.16.</li>
	<li>Image oocytes using a 40-63x objective on a confocal microscope, capturing &#60;1 &#181;m steps in the z-plane making sure to image the entire region of the metaphase spindle.</li>
	<li>To identify chromosome alignment status open image files using imaging software. 
	NOTE: The following steps describe this procedure using Image J (NIH) free downloadable software, however, alternate imaging software can be used.</li>
	<li>Drag image into image J control panel.</li>
	<li>In the opened z-series, use the point tool (available by clicking the point tool icon on the Image J control bar) to mark points at the end of each spindle pole in their respective z-slice by placing the tool over the spindle pole in the image and clicking on the image. Add these points to the Region of interest (ROI) manager by pressing Command+t on the keyboard.</li>
	<li>Determine the specific coordinates for each of the positions set in step 3.10 by highlighting the specific point in the ROI manager and clicking the measure button. This will provide a results table-containing x and y coordinates for each point. These will be the X1, Y1, and X1, Y2, points respectively.</li>
	<li>Next, determine the true Z coordinate. This is done by multiplying the slice number of the specific z-slice in the z-stack in which the spindle pole was identified (i.e. slice #2 of 6) that each individual point is in by the stack thickness (i.e. 1 &#181;m) (example: slice 2 x 1 &#181;m = 2). These will be the Z1 and Z2 points respectively.</li>
	<li>Determine spindle length using the Pythagorean theorem equation (a2 + b2 = c2) using the previously defined x, y, and z coordinates from steps 3.11-3.13. For each spindle the final length is equal to: 
	&#8730;((X1-X2)2+(Y1-Y2)2+(Z1-Z2)2)</li>
	<li>Determine the spindle midzone. This is calculated as one half the length of the spindle. Using the line tool in Image J, by clicking the line icon in the Image J tool bar, draw a line that is from one spindle pole to the spindle midzone. The length will be displayed on the Image J tool bar.</li>
	<li>Determine chromosome alignment status by assessing chromosome distance from the spindle midzone using the line measurement tool. Using the line tool available by clicking the line icon in the Image J tool bar, draw a line from the spindle midzone to the chromosome. The length will be displayed on the Image J tool bar. Chromosomes greater than 4 &#181;m from the spindle midzone are considered unaligned 18.</li>
</ol>

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The authors have nothing to disclose.

Because of the rapid and increasing identification of human genetic variants associated with disease, it is essential that systems are established to evaluate their biological significance. Understanding protein function in human meiosis poses particular challenges because human oocytes are precious and rare and human sperm are not amenable to genetic manipulation. Mouse oocytes are a mammalian model system valuable for evaluating human meiotic gene function 10,23</sup...

Infertility is a condition that affects 10-15% of the human population of reproductive age 1, from which nearly one-half seek medical treatment 2. Although the etiology of infertility is diverse and in many cases multifactorial, the most common genetic abnormality in humans is embryonic aneuploidy 3. Aneuploidy is defined as the deviation (either gain or loss) of the correct number of chromosomes in a cell. The phenomenon of aneuploidy in human embryos is common and increases with advanced maternal age 4,5. Four randomized controlled trials have highlighted the benefit of selecting only chromosomally normal (euploid) embryos for uterine transfer because this strategy resulted in increased implantation rates, lower miscarriage rates and a shorter time for achieving pregnancy 6,7,8,9. Therefore, understanding the etiology of human aneuploidy can have important implications in assisted reproduction.
Although pre-implantation genetic testing for aneuploidies is beneficial in infertility treatments, a thorough understanding of how aneuploidies originate is still lacking. It is widely accepted that there is a positive correlation of meiotic aneuploidies (originated during gamete production) and maternal age, however, some women present embryonic aneuploidy rates that deviate from the mean rate for their given age 4. These cases suggest that age alone is not always predictive of the risk of generating an aneuploid embryo. Other factors may play a role in increasing the risk of embryonic aneuploidy, such as gene variants.
A key aspect of investigating the potential contribution of a gene variant to aneuploidy during oocyte meiosis is to design a system to rapidly evaluate meiotic gene function. Due to ethical constraints and limited access, it is impractical to perform these experiments using human eggs. These issues can be circumvented by using mouse oocytes, and here a series of assays to assess human gene function during meiosis I are described. By microinjecting the messenger RNA (mRNA) coding for the gene variant of interest, the localization of the human protein in the mouse egg can be visualized and used to determine if the ectopic expression of the wild-type and mutated human protein results in any phenotypic alterations that could lead to aneuploidy. These phenotypes include an increase in microtubules that attach to the improper to sister kinetochore and the inability to support chromosome alignment at metaphase of meiosis I. Importantly, this protocol can be used to investigate both gain and loss of function genetic variants by establishing specific experimental conditions to challenge key events in oocyte meiosis such as spindle building and chromosome alignment 10.

<table>
	<tbody>
		<tr>
			<td>0.2 mL Seal-Rite PCR tube</td>
			<td>USA Scientific</td>
			<td>1602-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>1 kb DNA Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0313</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0243</td>
			<td></td>
		</tr>
		<tr>
			<td>6X DNA loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>9-well glass spot plate</td>
			<td>Thomas Scientific</td>
			<td>7812G17</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A3294&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-fluor-568 conjugated anti-mouse IgG</td>
			<td>Thermo Scientific</td>
			<td>A21050</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa-fluor-633 conjugated anti-human IgG</td>
			<td>Thermo Scientific</td>
			<td>A21091</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>VWR</td>
			<td>AA0356</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-vibration table</td>
			<td>Technical Manufacturing Corp</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Anti-Acetylated Tubulin antibody</td>
			<td>Sigma Aldrich</td>
			<td>T7451</td>
			<td>1:100 diution</td>
		</tr>
		<tr>
			<td>Anti-centromeric (CREST) antibody</td>
			<td>Antibodies Incorportated</td>
			<td>15-234</td>
			<td>1:30 dilution&#160;</td>
		</tr>
		<tr>
			<td>Barrier (Filter) Pipette tips</td>
			<td>Thermo Scientific</td>
			<td>AM12635</td>
			<td>Make sure compatable with your brand of pipettors. These are compatible with Eppendorf brand pipettors.&#160;</td>
		</tr>
		<tr>
			<td>BD Difco Dehydrated Culture Media: LB Agar, Miller (Luria Bertani)</td>
			<td>Fisher Scientific</td>
			<td>DF0445-07-6</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria Bertani)</td>
			<td>Fisher Scientific</td>
			<td>DF0446-07-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tubing</td>
			<td>Sutter</td>
			<td>B100-75-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Center Well organ culture dish</td>
			<td>VWR</td>
			<td>25381-141</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td></td>
			<td></td>
			<td>For incubator</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Centrifuge (With cooling ability)</td>
			<td>Thomas Scientific</td>
			<td></td>
			<td>any standard model&#160;</td>
		</tr>
		<tr>
			<td>Cover Glass 11 x 22 mm</td>
			<td>Thomas Scientific</td>
			<td>6663F10</td>
			<td></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>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC H20</td>
			<td>Life Technologies</td>
			<td>AM9922</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Dry Bath</td>
			<td>Thermo Scientific</td>
			<td>888700001</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy A high fidelity cloning enzyme</td>
			<td>Agilent</td>
			<td>600400</td>
			<td>For DNA cloning&#160;</td>
		</tr>
		<tr>
			<td>Enzymes for linearizing pIVT</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>NdeI or KasI can be used</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Thermo Scientific</td>
			<td>155585011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope&#160;</td>
			<td></td>
			<td></td>
			<td>Any Fluorescent microscope may be used</td>
		</tr>
		<tr>
			<td>Formaldehyde (37%)</td>
			<td>Thermo Fisher Scientifc</td>
			<td>9311</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde RNA loading dye</td>
			<td>Ambion</td>
			<td>8552</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides (Uncharged) 25X75 mm</td>
			<td>Fisher Scientific</td>
			<td>12-544-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Full Length cDNA Clones</td>
			<td></td>
			<td></td>
			<td>Can be obtained from any vendor that supplies open reading frame clones</td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Globin Forward Primer for pIVT Construct</td>
			<td></td>
			<td></td>
			<td>5&#39;- GAA GCT CAG AAT AAA CGC -3&#39;.&#160; Can be purchased from any company that generates custom oligonucelotides</td>
		</tr>
		<tr>
			<td>Globin Reverse Primer for pIVT Construct</td>
			<td></td>
			<td></td>
			<td>5&#39;- ATT CGG GTG TTC TTG AGG CTG G -3&#39; Can be purchased from any company that generates custom oligonucelotides</td>
		</tr>
		<tr>
			<td>Holding pipettes</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td>Vacutip</td>
		</tr>
		<tr>
			<td>Humidified Chamber</td>
			<td></td>
			<td></td>
			<td>Tupperware can be used</td>
		</tr>
		<tr>
			<td>Illustra Ready-To-Go RT-PCR beads</td>
			<td>GE Life Sciences</td>
			<td>27925901</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>any standard model with CO2 and water jacketed technology</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon instruments</td>
			<td></td>
			<td>Any Standard model</td>
		</tr>
		<tr>
			<td>Image J (NIH) Software</td>
			<td>NIH</td>
			<td></td>
			<td>Image Analysis software</td>
		</tr>
		<tr>
			<td>Lid of 96 well plate</td>
			<td>Nalgene Nunc International</td>
			<td>263339</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Adhesion 0.5 mL microcentrifgue tube</td>
			<td>USA Scientific</td>
			<td>1405-2600</td>
			<td></td>
		</tr>
		<tr>
			<td>MacVector&#160;</td>
			<td>MacVector</td>
			<td></td>
			<td>Sequence analysis software</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Selleckchem</td>
			<td>S2619</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Millenium RNA Markers-Formaldehyde&#160;</td>
			<td>Ambion</td>
			<td>AM7151</td>
			<td></td>
		</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 used embryo-tested, sterile-filtered</td>
		</tr>
		<tr>
			<td>Monastrol</td>
			<td>Sigma-Aldrich</td>
			<td>M8515</td>
			<td>Resuspend in DMSO at 100 mM</td>
		</tr>
		<tr>
			<td>Mouthpiece</td>
			<td>Biodiseno</td>
			<td>MP-001-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 tank</td>
			<td></td>
			<td></td>
			<td>for antivibration table</td>
		</tr>
		<tr>
			<td>Nail Polish; Clear</td>
			<td></td>
			<td></td>
			<td>Any clear nailpolish can be used</td>
		</tr>
		<tr>
			<td>NanoDrop Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>NorthernMax 10X Denaturing Gel Buffer</td>
			<td>Life Technologies</td>
			<td>AM8676</td>
			<td></td>
		</tr>
		<tr>
			<td>NorthernMax 10X Running buffer</td>
			<td>Life Technologies</td>
			<td>AM8671</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MOPS SDS Running buffer</td>
			<td>Thermo Scnentific</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ Culture Dish 60x15mm</td>
			<td>Life Technologies</td>
			<td>08-772-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Polysciences, Inc.&#160;</td>
			<td>577773</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>4484075</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 139 mm</td>
			<td>Thermo Fisher Scientifc</td>
			<td>501V</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35 mm</td>
			<td>Thermo Fisher Scientifc</td>
			<td>121V</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 60 mm</td>
			<td>Falcon BD</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Picoinjector</td>
			<td>XenoWorks Digital Microinjector</td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Flaming-Brown Micropipette puller</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>pIVT plasmid</td>
			<td>AddGene</td>
			<td>32374</td>
			<td>Empty vector suitable for oocyte expression.</td>
		</tr>
		<tr>
			<td>Pregnant Mare Serum Gonadotropin</td>
			<td>Lee BioSolutions</td>
			<td>493-10</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>purification of up to 20 uL of plasmid DNA</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Quikchange II site directed mutagenesis kit</td>
			<td>Agilent&#160;</td>
			<td>200523</td>
			<td>mutagenesis kit for insertions and deletions</td>
		</tr>
		<tr>
			<td>Quikchange lightning multi-site directed mutagenesis kit</td>
			<td>Agilent&#160;</td>
			<td>210512</td>
			<td>mutagenesis kit for single site changes</td>
		</tr>
		<tr>
			<td>Scissors (Fine point)</td>
			<td>Fine science tools</td>
			<td>14393</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (Medium point)</td>
			<td>Fine science tools</td>
			<td>WP114225</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Warmer</td>
			<td></td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Spectrophotometer (Nanodrop)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td>any standard model</td>
		</tr>
		<tr>
			<td>Subcloning Efficiency DH5a Competent Cells</td>
			<td>Thermo Fisher Scientifc</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Bioscienes</td>
			<td>309623</td>
			<td>1 ml, 27G(1/2)</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 mMessage Machine high-yield capped RNA transcription kit</td>
			<td>Life Technologies</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma-Aldrich</td>
			<td>x-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer (Fine point- Size 5)</td>
			<td>Fine science tools</td>
			<td>SN.743.12.1</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Dnase/Rnase-Free Distilled Water</td>
			<td>Thermo Fisher Scientifc</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium Bromide 10mg/mL</td>
			<td>Thermo Fisher Scientifc</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP UV/White lite transilluminator</td>
			<td>Fisher Scientific</td>
			<td>UV95041501</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

using mouse oocytes to assess human gene function during meiosis i

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

After in vitro transcription high quality RNA will have an A260/A280 ratio of (1.8-2.2) and an A260/A230 ratio &#8805;1.7 when measured using a spectrophotometer. The picture in Figure 1 shows the migration of in vitro-produced RNA on a denaturing agarose gel after electrophoresis. A band that is smeared in pattern or a sample that has multiple sized bands can indicate contamination or degradation of the sample. Alternatively, multiple bands...

Watch this Scientific Journal Video about Using Mouse Oocytes to Assess Human Gene Function During Meiosis I at JoVE.com

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

using-mouse-oocytes-to-assess-human-gene-function-during-meiosis-i

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Unit 2

Unit 1

Meiosis

SCIENTISTS IN ACTION

8.8K Views.  IVI-RMA New Jersey. As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Video: Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...