Name: generation of genetically modified mice through the microinjection of oocytes
Uploaded: 2017-06-15T13:00:00
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

The authors thank the staff of the animal facility (BRC) for their ongoing support. This work was funded by the National Health and Medical Research Council and the Australian Research Council.

All procedures have been approved by the University of New South Wales Animals Care and Ethics Committee.
1. Preparation of the Transgene (Random Integration)
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	<li>Analytical agarose gel electrophoresis.
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		<li>Digest the plasmid to excise the transgene using appropriate enzymes (1 h incubation) or fast-digest enzymes (15 to 30 min incubation) in a thermocycler following the manufacturer&#39;s recommendations (see Figure 2A and its leg...

<ol>
	<li>Ittner, L. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+estrous+cycle+identification+tool+and+images.">Mouse estrous cycle identification tool and images.</a> PLoS One. 7 (4), e35538 (2012).</li><li>Whitten, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+oestrous+cycle+of+the+mouse+by+external+stimuli+associated+with+the+male.">Modification of the oestrous cycle of the mouse by external stimuli associated with the male.</a> J Endocrinol. 13 (4), 399-404 (1956).</li><li>Ye, S., Dhillon, S., Ke, X., Collins, A. R., Day, I. 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Critical steps within the protocol
The generation of genetically modified mice is known to be technically challenging. However, the protocol presented here is an optimized and simplified method that allows one to master and troubleshoot the technique in record time. There are two steps necessary for the successful completion of the technique. First, the synthesis of linear DNA templates (for the synthesis of sgRNAs) can be achieved without magnesium chloride (MgCl2). However, it is hig...

Animal models, both in vertebrates and invertebrates, have been instrumental to examining the pathophysiology of human conditions such as Alzheimer&#39;s disease1,2. They are also invaluable tools to search for disease modifiers and to ultimately develop novel treatment strategies in the hope of a cure. Although each model has intrinsic limitations, the use of animals as entire systemic models is vital to biomedical research. This is because the metabolic and complex physiological environment cannot be entirely simulated in tissue culture.
To date, the mouse remains the most common mammalian species used for genetic manipulation because it features several advantages. The physiological processes and genes associated with diseases are highly conserved between mice and humans. The mouse was the first mammal to have its full genome sequenced (2002), one year before the human genome (2003). Aside from this wealth of genetic information, the mouse has good breeding capacities, a fast development cycle (6 weeks from fertilization to weaning), and a reasonable size. All these advantages, coupled with physiological indicators, such as distinct coat colors (required for crossing strategies), made the mouse an attractive model for genetic manipulation. Notably, in the very early age of modern genetics, Gregor Mendel started working on mice before moving to plants3.
Gene transfer techniques resulted in the generation of the first transgenic mouse over three decades ago4, initially created using viral delivery. However, researchers soon realized that one of the main challenges of mouse transgenesis was the inability to control the fate of the exogenous DNA. Because the viral delivery of transgenes into mouse oocytes resulted in multiple copies integrated randomly into the genome, the possibility of establishing subsequent transgenic lines was limited.
One such limitation was overcome when Gordon et al. generated the first transgenic mouse line by microinjection5,6. This began the era of recombinant DNA technology, and the parameters influencing the outcome of a microinjection session have been widely studied7. Although microinjection does not allow for control over the integration site of the transgene (which eventually results in specific expression levels for each founder mouse), the main advantage of pronuclear microinjection remains the formation of concatemers (i.e., arrays of multiple copies of the transgene, linked in series) before genomic integration5. This characteristic has been used over the years to establish thousands of transgenic mouse lines that overexpress a gene of interest. Since then, transgenesis, the artificial modification of an organism&#39;s genome, has been extensively used to identify the role of single genes in the occurrence of diseases.
A further key achievement in manipulating the mouse genome was reached when Mario Capecchi successfully disrupted a single gene in the mouse, opening the era of gene targeting8. However, major drawbacks quickly emerged from ES cell-based gene targeting, including the challenges of culturing ES cells, the somewhat variable degree of chimerism, and the length of the process (i.e., 12-18 months, minimum, to obtain the mouse).
Recently, advances in new technologies, such as engineered endonucleases (e.g., zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9)) have emerged as alternative methods to accelerate the process of gene targeting in mice9,10. These endonucleases can readily be injected into mouse oocytes by microinjection, allowing for the generation of gene-targeted mice in as little as 6 weeks.
Since the first report on the use of CRISPR for genome editing11, this bacterial adaptive immune system has superseded ZFN and TALEN because of its many advantages, including the ease of synthesis and the ability to target multiple loci at once (referred to as &#34;multiplexing&#34;). CRISPR was first used for gene targeting in mice12 and has since been applied to countless species, from plants to humans13,14. To date, there is no report of a single species resistant to CRISPR genome editing.
The two main limiting steps of the generation of transgenic mice are the injection of oocytes and the reimplantation of these oocytes into pseudo-pregnant females. Although this technique has been described by us15 and others16, recent technical improvements in mouse embryology and gene transfer techniques have revolutionized the process of generating genetically modified mice. These improvements will be described herein.

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			<td height="21" style="height:21px;width:307px;">Micropipette 0.1-2.5 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">4920000016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 2-20 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 20-200 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 100-1000 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular weight marker&#160;</td>
			<td>Bioline</td>
			<td>BIO-33025</td>
			<td>HyperLadder 1kb</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular weight marker&#160;</td>
			<td>Bioline</td>
			<td>BIO-33056</td>
			<td>HyperLadder 100 bp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">EDTA buffer</td>
			<td>Sigma-Aldrich</td>
			<td>93296</td>
			<td>10x - Dilute to 1x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Ethidium bromide</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15585011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Safe gel stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Gel extraction kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">28706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">PCR purification kit (Qiaquick)</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vacuum system (Manifold)</td>
			<td style="width:147px;">Promega</td>
			<td style="width:119px;">A7231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-free microinjection buffer&#160;</td>
			<td>Millipore</td>
			<td>MR-095-10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrafree-MC microcentrifuge filter&#160;</td>
			<td>Millipore</td>
			<td>UFC30GV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cas9 mRNA</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">CAS9MRNA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">CRISPR expressing plasmid (px330)</td>
			<td style="width:147px;">Addgene</td>
			<td style="width:119px;">42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nuclease free water</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Phusion polymerase</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Quick High Yield RNA kit</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>E2050S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">RNA purification spin columns (NucAway)</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td>AM10070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">ssOligos</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>OLIGO STANDARD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Donor plasmid</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">GeneArt</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Hyaluronidase</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">KSOMaa embryo culture medium</td>
			<td>Zenith Biotech&#160;</td>
			<td>ZEKS-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mineral oil</td>
			<td>Zenith Biotech&#160;</td>
			<td>ZSCO-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">M2 Medium</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>M7167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytochalasin B</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>C6762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mouthpiece</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Glass microcapillaries</td>
			<td style="width:147px;">Sutter Instrument</td>
			<td style="width:119px;">BF100-78-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Proteinase K</td>
			<td>Applichem</td>
			<td>A3830.0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Dumont #5 forceps</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Iris scissors</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vessel clamp</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">18374-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Wound clips&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">12040-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Clips applier&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">12018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cauterizer</td>
			<td>Fine Science Tools&#160;</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-absorbable surgical sutures (Ethilon 3-0)</td>
			<td>Ethicon</td>
			<td>1691H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">5% CO2 incubator</td>
			<td style="width:147px;">MG Scientific</td>
			<td style="width:119px;">Galaxy 14S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Spectrophotometer</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Nanodrop 2000c</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermocycler</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">6321 000.515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Electrophoresis set up</td>
			<td style="width:147px;">BioRad</td>
			<td style="width:119px;">1640300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">UV Transilluminator</td>
			<td style="width:147px;">BioRad</td>
			<td style="width:119px;">1708110EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermocycler</td>
			<td style="width:147px;">Eppendorf</td>
			<td>6334000069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscopic microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Inverted microscope</td>
			<td>Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">2x Micromanipulators</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5188000.012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Oocytes manipulator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5176000.025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microinjector (Femtojet)</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5247000.013</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Mice C57BL/6J strain</td>
			<td style="width:147px;">Australian BioResources</td>
			<td>C57BL/6JAusb&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of genetically modified mice through the microinjection of oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

Below, the workflows for microinjection in the case of random integration and CRISPR-mediated gene targeting are described (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55765/55765fig1v2.jpg" /> 
Figure 1: Typical Workflow for the Generation of Genetically Modified Mice. For random integration, the purified transgene is injected into the pronucleus of f...

Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

The overall goal of this protocol, is to describe the procedures required for the successful generation of genetically modified mice by microinjection of oocytes. This protocol can help researchers and technical staff involved either in the field of mouse genesis, but also in gene targeting. The main advantage of this technique, is that it relies on the direct injection of mouse oocytes, both for random integration, but also for gene targeting, allowing the generation of genetically modified mice in as little as six weeks.To prepare a single guide RNA, after selecting the desired two target sequences and making primers according to the text protocol, synthesis a linear DNA template by diluting the PX330 plasmid to 10 nanograms per microliter in nuclease free water. Prepare a master mix, adding the polymerase at the end, and keep on ice. Then mix and split the solution into eight PCR tubes.Add one microliter of PXR330 per tube, and run the following program. Next, use a PCR purification kit, a manifold and a vacuum source to purify the PCR product. Add five volumes of binding buffer to one volume of the PCR sample and mix.Place the silicon membrane spin column on the manifold. To bind DNA, apply all eight samples to the column, in vacuum. To wash the sample, add 0.75 milliliters of wash buffer to the column and vacuum.Then, centrifuge the column at 12, 000 times G for 60 seconds to eliminate residual ethanol. Place the column in a clean 1.5 milliliter micro centrifuge tube, then, to elute the DNA, add 30 microliters of nuclease free water to the center of the membrane. Let the column stand for one minute and centrifuge it, 12, 000 times G for 60 seconds.Then use a spectrophotometer to measure the DNA concentration. To carry out in vitro transcription using a t7 RNA synthesis kit, prepare the master mix and incubate the reaction in a thermo cycler at 37 degree celsius for three hours. Add 28 microliters of nuclease free water and two microliters of DNAse one, and incubate the reaction for another 15 minutes.To purify the RNA, dissolve the powder contained in the provided spin column and 650 microliters of nuclease free micro injection buffer. Carefully removing all the air bubbles, cap the tube and hydrate the solution at room temperature for five to 15 minutes. Remove the blue cap at the bottom and place the column in a two milliliter tube.Then centrifuge the tube at 750 times G at room temperature for two minutes. Then place the column in a fresh 1.5 milliliter tube and apply 50 microliters of the RNA solution drop wise to the center, without touching the column wall. Then, spin the column at 750 times G for two minutes.Measure the RNA concentration and assess the quality of the RNA according to the text protocol. Using nuclease free micro injection buffer, dilute the case nine mRNA to 50 nanograms per microliter and the sgRNAs to 12.5 nanograms per microliter for gene knockout experiments. Add the donor template at a concentration of 200 nanograms per microliter, for genome editing based on homology direct repair and keep it on ice.Use the aspirator mouth piece, to wash the oocytes with four fresh drops of casein amino acids. And transfer them to a final drop of casome eight medium, overlaid with mineral oil. To carry out pro nuclear injection for random integration, under the stereo microscope, use the aspirator mouth piece to transform approximately 50 eggs to a drop of m2 medium, overlaid with mineral oil that will be used as the injection chamber.Transfer the injection chamber to an inverted microscope and inject the fertilized oocytes with a few picoliters of the injection mix. The pronucleus will swell if the injection is successful. Perform cytoplasmic injection for gene targeting, use a mouthpiece to transfer approximately 50 eggs to a drop of casome A, containing five micrograms per milliliter cytochalasin B and incubate the eggs at 37 degree celsius and 5%carbon dioxide for five minutes.Transfer the eggs to the injection chamber, then, at very low pressure, inject a few picoliters of the injection mix into the cytoplasm. Using the compensation pressure of the automated microinjector where possible. After the injection, use the mouthpiece to transfer the oocytes back into a drop of casomeamino acids.Keep them at 37 degree celsius and 5%carbon dioxide until they are loaded for re-implantation into the oviduct of the pseudo pregnant females. After anesthetizing a plugged female mouse according to the text protocol, to carry out re-implantation under sterile conditions, expose the reproductive tract by using a scalpel to a one centimeter long incision parallel to the dorsal mid line. Then, with scissors, cut the muscle and use forceps to grab the fat pad.Gently pull the ovary out, until its attached oviduct and uterus are clearly visible. Then use a vessel clamp to fix the fat pad. Under the stereo microscope, use a pair of micro scissors to make an incision into the wall of the oviduct, a few millimeters upstream of the ampulla.Also, under the stereo microscope, load 25 micro injected eggs into the glass capillary connected to the mouthpiece. Then, insert the glass capillary into the oviduct and expel the eggs until an air bubble is visible inside the ampulla. To ensure that the re-implantation has been successful, you must check for the presence of the air bubble in the ampulla, which guarantees that all the eggs have been expelled at the right location and none remain in the glass capillary.Gently remove the glass capillary and place the reproductive tract back into the abdomen. Then use three zero, non-absorbable surgical sutures to suture the incision and then, use wound clips to close the skin. Place the mouse on a warm mat to avoid hypothermia, and inject analgesic subcutaneously.Monitor the mouse until a full recovery. Finally, carry out genome typing and sequencing according to the text protocol. This figure shows analytical gels demonstrating the quality of the transgene purification, and the sgRNA synthesis which are critical to successfully generate genetically modified mice.Shown here, is a typical read out of a micro injection session for random integration. The genotyping primer should sit on the transgene and should be designed to generate a fragment of 200 to 800 base pairs. In this read out for gene targeting, the primers for selected to hybridize with the genomic DNA outside of the region eventually excised by the two guys.This strategy allows for the direct identification of heterozygous and homozygous knock out founders. As illustrated here, when each step of the protocol is optimized, the percentage of transgenic pup should reach 10%to 25%for random integration, which is somewhat low compared to the ability of the CRISPR components to edit the mouse genome. Using CRISPR for gene targeting, the number of founders is generally higher, ranging from 25%to 100%it is not uncommon to obtain an entire progeny that is successfully homozygous when edited using CRISPR.After its development, this technique allows researchers in fields such as, neuroscience or cancer for instance, to use new mask models to discover pathological mechanisms and eventually translate this into therapeutic strategies.

Research

JoVE Journal

Developmental Biology

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Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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