Name: production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex
Uploaded: 2018-01-09T12:30:00
Description: Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Research reported in this publication was supported by the National Institutes of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health under award number 1R41OD021979 (to ZW) and by a research grant from the Next-Generation BioGreen 21 Program, Republic of Korea, grant no. PJ01107704 (to ZW) and grant no. PJ01107703 (to IK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or BioGreen 21. We thank Dr. Nikolas Robl for editing the manuscript.

The procedures described in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of Utah State University (IACUC protocol: 2484). Hamsters used in this protocol are adult (6 - 10 weeks of age) LVG strain golden Syrian hamsters. All hamsters are housed in the vivarium at the Bioinnovation center, Utah State University. Room temperature is set at 23 &#176;C, humidity is set at 40 - 50%, and light cycle is set 14L:10D (light:dark). Whenever possible, embryo manipulation and surugical proced...

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To better exploit the potential of golden Syrian hamsters as models of human disease, we developed a PN injection protocol for delivering a CRISPR/Cas9 complex to target the hamster genome. The PN injection protocol optimizes several key variables including the embryo culture medium, temperature, and wavelengths of light13. There are also several hamster-specific animal handling procedures that need to follow for successfully conducting gene targeting in the hamster. For example, sexually matured ...

The golden Syrian hamster (Mesocricetus auratus) is one of the most widely used rodents for biomedical research. According to the U.S. Department of Agriculture, approximately 100,000 hamsters were used in the United States in 2015, representing 13% of total laboratory animal usage among the species covered by the Animal Welfare Act (http://www.aphis.usda.gov; accessed March 10, 2017).
The hamster offers several advantages over other rodents in the study of a number of human diseases. For example, the histopathology of N-nitrosobis(2-oxopropyl)amine (BOP) induced pancreatic ductal adenocarcinomas in hamster is similar to human pancreatic tumors, while BOP treatment mainly induces thyroid gland tumors in rats and lung and liver tumors in mice1. Because hamsters are the only small rodent found to support the replication of adenoviruses, they are also the model of choice for testing adenovirus-based oncolytic vectors and anti-adenovirus drugs2,3,4. Another example wherein the hamster model offers an advantage over mice and rats is in the study of hyperlipidemia. Humans and hamsters exhibit great similarities in lipid metabolic pathways and both species carry the gene encoding cholesteryl ester transfer protein (CETP), which plays a central role in lipid metabolisms, while CETP is absent in mice and rats5. Additionally, hamsters develop hemorrhagic disease more representative of the human manifestation following exposure to Ebola virus6. Hamsters are also the models of choice for studying atherosclerosis7, oral carcinomas8, and inflammatory myopathies9. Recently, it has also been demonstrated that hamsters are highly susceptible to Andes virus infection and develop hantavirus pulmonary syndrome-like disease, providing the only rodent model of Andes virus infection10.
To address the unmet need for novel genetic animal models to study the human diseases where no reliable small rodent model is available, we recently have succeeded in applying the CRISPR/Cas9 system to the hamster and have produced several lines of genetically engineered hamsters11. Hamster zygotes are highly sensitive to environmental milieus such that the PN injection protocols developed in other species are unsuitable. Therefore, we developed a PN injection protocol for the hamster that accommodates the special requirements for handling hamster embryos in vitro. Here, we describe the detailed PN injection procedure using the CRISPR/Cas9 system and the accompanying steps, from the preparation of single guide RNA (sgRNA) to the transfer of injected embryos into recipient females.

<table>
	<tbody>
		<tr>
			<td>Cas9</td>
			<td>Invitrogen</td>
			<td>B25640</td>
			<td>1 ug/ul (~6.1 uM)</td>
		</tr>
		<tr>
			<td>GeneArtTM Precision Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>A29377</td>
			<td>For sgRNA synthesis</td>
		</tr>
		<tr>
			<td>Albumin from human serum</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td>For cultivation medium</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>NI-150</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>New Brunswick</td>
			<td>Galaxy 14S</td>
			<td>For embryo cultivation</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>PB-7</td>
			<td>For making injection needles</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti-S</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>invitrogen</td>
			<td>SMZ745T</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M1840</td>
			<td>Keep in dark</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4877-2000IU</td>
			<td>For superovulation</td>
		</tr>
	</tbody>
</table>

production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

The efficiency of the described protocol in producing genetically modified hamsters depends on the outcomes of the following two critical steps: the live birth rate of recipient females and the number of live pups with the intended genetic modifications. The live birth rate is a direct results of the embryo quality and the skill of the individual performing the PN injection and embryo transfer procedures. To ensure that the developmental potential of manipulated embryos is not compromised...

Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

The overall goal of this protocol is to provided viewers with detailed procedures to produce Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex. This PN injection protocol we developed in the Golden Syrian Hamster makes genetic engineering in this business possible. This technology is a great contribution to the field of animal model development.Due towards extreme sensitivity of hamster embryos to the environment, we developed a unique pronuclear injection protocol to accommodate the special niche of hamster embryos. The development of this technique is a great step forward in human disease modeling, because for the first time, genetically engineered hamster models can be produced. For this experiment, one day prior to pronuclear or PN injection, prepare Zygote handling dishes and PN injection drop in a lid of a 35 millimeter dish with HECM-9 medium.Cover the medium drops with mineral oil. To balance the drops and HECM-9 medium, incubate it at 37.5 degrees celsius overnight. On the day of PN injection, prepare embryo-flushing dishes using HECM-9.Store them in the incubator until needed. To isolate oviducts, place a euthanized superovulated female hamster in dorsal recumbency on a surgical drape. Scrub the abdomen with 70%ethanol.Next, firmly hold skin and abdominal muscle layer at the midline, and make an incision along the end of the abdomen. To expose the abdominal organs, incise the peritoneum. Next, use a fine forceps to grasp one of the uterine horns, and retract the tissue from the abdomen.Separate the uterus from the mesometrium and fat tissue. Make the first cut between the oviduct and ovary and a second next the oviduct-uterus junction while including a small part of the uterus. Place the oviducts in the same flesh dish.To collect zygotes under the dissection microscope, use a number five Dumont forceps to grasp the side of the uterine horn. Insert a homemade needle into the lumen of the oviduct from the infundibular end. Then, flush the oviduct with 300 to 400 microliters of HECM-9 medium.Immediately after flushing, collect the zygotes;and wash them twice with HECM-9 in the handling dish. All zygotes should be denuded from the cumulous cells at this stage of development. Right after that, place the zygotes in an incubator until the injection to avoid exposure to light.To prepare for injection, thaw the Cas9-sgRNA RNPs on ice. Immediately before the injection, load the injection needle with RNP solution by placing the rear end of the needle into the tube containing the solution allowing it to fill the needle by capillary action Next, fill the holder pipette with mineral oil up to approximately three millimeters of the needle bend. Place the holder in the micro injector with the tip of the holder pipette horizontal to the bottom of the dish.Fill the needle tip with medium by suction. Then, set the injection needle at a 10 to 15 degree angle opposite to the holder. Next, identify a zygote, and use the holder needle to apply a fine suction.Male, which is slightly bigger, and female pronuclei should preferably be within the same focal range and aligned parallel to the holder. Using the injection needle at a three o'clock position, penetrate the zona pellucida and male pronuclei. Once the pronucleus swells, withdraw the injection needle quickly to prevent the nucleus from adhering to the needle and to avoid damaging it.Efficiency of gene targeting for STAT2 and RAG1 genes by the Cas9/sgRNA system is evaluated by live birth rate of recipient females and the number of live pups with the intended genetic modifications. Genotyping results from a PCR restriction fragment length polymorphism assay, or PCR-RFLP, of pups, show the efficiency of gene targeting. Lane one indicated biallelically targeted alleles, and lanes three and seven show monoallelically, or mosaically, targeted alleles.This technology has paved the way for researchers to develop newer genetically engineered hamster models of human disease. After watching this video, you should have a good understanding of how to produce genetically engineered Golden Syria Hamsters by a pronuclear injection of the CRISPR/Cas9 Complex.

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