Name: generation of transgenic rats using a lentiviral vector approach
Uploaded: 2020-05-17T12:30:00
Description: Watch this Scientific Journal Video about Generation of Transgenic Rats using a Lentiviral Vector Approach at JoVE.com

This study was supported by the ANIMOD project within the Team Tech Core Facility Plus program of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development Fund to WK.

The production and application of viral vectors was in accordance with Biosafety Level 2 guidelines and was approved by the Polish Ministry of Environment. All experimental animal procedures that are described below were approved by the Local Ethical Committee. The animals were housed in individually ventilated cages at a stable temperature (21&#8211;23 &#176;C) and humidity (50&#8211;60%) with ad libitum access to water and food under a 12 h/12 h light/dark cycle.
1. Lentiviral vector productio...

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Advances in transgenic technologies have made rodent models an invaluable tool in biomedical research. They provide the opportunity to study genotype-phenotype relationships in vivo. Here, we present a widely available alternative for conventional transgenesis by pronuclear injections. The use of lentiviral gene transduction bypasses the need for demanding microinjections because viral vectors can be injected under the zona pellucida. This approach does not affect embryo integrity, which essentially guarantees a 100% sur...

For many years, laboratory rodents, such as rats and mice, have been used to model human physiological and pathological conditions. Animal research has led to discoveries that were unattainable by any other means. Initially, genetic studies focused on the analysis of spontaneously occurring disorders and phenotypes that are considered to closely mimic the human condition1. The development of genetic engineering methods allowed the introduction or deletion of specific genes to obtain a desired phenotype. Therefore, the generation of transgenic animals is recognized as a fundamental technique in modern research that allows studies of gene function in living organisms.
Transgenic animal technology has become possible through a combination of achievements in experimental embryology and molecular biology. In the 1960s, the Polish embryologist A. K. Tarkowski published the first work on mouse embryo manipulation during the early stages of development2. Additionally, molecular biologists developed techniques to generate DNA vectors (i.e., carriers) for the inter alia introduction of foreign DNA into the animal&#8217;s genome. These vectors allow the propagation of selected genes and their appropriate modification, depending on the type of research that is conducted. The term &#8220;transgenic animal&#8221; was introduced by Gordon and Ruddle3.
The first widely accepted species that was used in neurobiology, physiology, pharmacology, toxicology, and many other fields of biological and medical sciences was the Norway rat, Rattus norvegicus4. However, because of the difficulty in manipulating rat embryos, the house mouse Mus musculus has become the dominant animal species in genetic research5. Another reason for the primacy of the mouse in such research was the availability of embryonic stem cell technology to generate knockout animals for this species. The most commonly used technique of transgenesis (2&#8211;10% of transgenic offspring relative to all born animals) is the microinjection of DNA fragments into a pronucleus of a fertilized oocyte. In 1990, this approach, which was first introduced in mice, was adapted for rats6,7. Rat transgenesis by pronuclear injection is characterized by lower efficiency8 compared with mice, which is strictly related to the presence of elastic plasma and pronuclear membranes9. Although the survival of embryos after manipulation is 40&#8211;50% lower than in mice, this technique is considered a standard in the generation of genetically modified rats10. Alternative approaches that can guarantee efficient transgene incorporation and higher survival rates of injected zygotes have been investigated.
The key determinant of stable transgene expression and transmission to progeny is its integration into the host cell genome. Lentiviruses (LVs) have the distinctive feature of being able to infect both dividing and non-dividing cells. Their use as a tool for the incorporation of heterologous genes into embryos proved to be highly efficient11, and transgenic individuals are characterized by stable expression of the incorporated DNA fragment. The efficacy of lentiviral vectors has been confirmed for the genetic modification of mice12,13, rats12,14, and other species11. In this method, the LV suspension is injected under the zona pellucida of the embryo at the stage of two pronuclei. This technique essentially guarantees 100% survival of the embryos because the oolemma remains unaffected. The production of high-quality and relatively highly concentrated LV suspensions are crucial factors. However, lower concentrations of LV suspensions can be overcome by repeated injections11, which increases the amount of viral particles at the egg surface while not affecting membrane integration. Embryos that are subjected to repeated injections into the perivitelline space develop further, and transgenic offspring can transmit the transgene through the germline. The efficiency of transgenic rat generation by lentiviral transgenesis can be as high as 80%12.
Here, we describe the production of HIV-1-derived recombinant lentivirus that was pseudotyped with vesicular stomatitis virus (VSV) G envelope protein. The use of the second-generation packaging system VSV pseudotype determines the wide infectivity of viral particles and allows the production of highly stable vectors that can be concentrated by ultracentrifugation and cryopreserved. After titer verification, the vectors are ready to be used as a vehicle for transgene delivery into albino Wistar rat zygotes. After a series of injections, the embryos can be cultured overnight and transferred at the two-cell stage to foster mothers. At this point, one of two alternative approaches can be considered. The standard procedure utilizes pseudopregnant females as embryo recipients. However, when the pregnancy rate is low after mating with vasectomized males, the embryos can be implanted into pregnant Wistar/Sprague-Dawley (SD) females that are mated with fertile male rats with a dark fur color (e.g., Brown Norway [BN] rats). The color of the fur allows the distinction of offspring from natural pregnancy from offspring that originate from the transferred manipulated embryos.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">7500 Real Time PCR System</td>
			<td style="width:186px;">Applied Biosystems</td>
			<td style="width:354px;"></td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Aerrane (isoflurane)</td>
			<td style="width:186px;">Baxter</td>
			<td style="width:354px;">FDG9623</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Atipam 5 mg/ml</td>
			<td>Eurovet Animal Health BV</td>
			<td>N/A</td>
			<td>0.5 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Baytril 25 mg/ml (enrofloksacin)</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td>5-10 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Borosilicate glass capillaries with filament GC100TF-15</td>
			<td style="width:186px;">Harvard Apparatus Limited</td>
			<td style="width:354px;">30-0039</td>
			<td style="width:172px;">injection capillary</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacaine 25 mg/ml</td>
			<td>Advanz Pharma</td>
			<td>N/A</td>
			<td>0.25% in&#160; 0.9% NaCl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Butomidor 10 mg/ml (butorphanol&#160; tartrate)</td>
			<td>Orion Pharma</td>
			<td>N/A</td>
			<td>1 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">CELLSTAR Tissue Cell Culture Dish 35-mm</td>
			<td style="width:186px;">Greiner Bio-One</td>
			<td style="width:354px;">627160</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:496px;">CELLSTAR Tissue Cell Culture Dish 60-mm</td>
			<td style="width:186px;">Greiner Bio-One</td>
			<td style="width:354px;">628160</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">CellTram Oil</td>
			<td style="width:186px;">Eppendorf</td>
			<td style="width:354px;">5176 000.025</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cepetor (Medetomidine) 1 mg/ml</td>
			<td>cp-pharma</td>
			<td>N/A</td>
			<td>0.5 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chorulon, Human Chorionic Gonadotrophin&#160;</td>
			<td>Intervet</td>
			<td>N/A</td>
			<td>150 IU/ ml ml 0.9% NaCl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">DMEM low glucose</td>
			<td style="width:186px;">Sigma Aldrich</td>
			<td style="width:354px;">D6048</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">DNase, RNase-free</td>
			<td style="width:186px;">A&#38;A Biotechnology</td>
			<td style="width:354px;">1009-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EmbryoMax Filtered Light Mineral Oil</td>
			<td>Sigma</td>
			<td>ES-005-C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Envelope protein coding plasmid for lentiviral vectors (VSVg plasmid)&#160;</td>
			<td style="width:186px;">ADDGENE</td>
			<td style="width:354px;">14888</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">FemtoJet</td>
			<td style="width:186px;">Eppendorf</td>
			<td style="width:354px;">4i /5252 000.013</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Fetal Bovine Serum</td>
			<td style="width:186px;">Sigma Aldrich</td>
			<td style="width:354px;">F9665-500ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Folligon, Pregnant Mare&#8217;s Serum Gonadotropin&#160;</td>
			<td>Intervet</td>
			<td>N/A</td>
			<td>125 IU/ml in .9% NaCl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEK 293T cells&#160;</td>
			<td style="width:186px;">ATCC</td>
			<td style="width:354px;">ATCC CRL-3216</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase from Bovine Testis&#160;</td>
			<td>Sigma</td>
			<td>H4272-30MG</td>
			<td>0.5 mg/ml in M2 medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Inverted Microscope&#160;</td>
			<td style="width:186px;">Zeiss</td>
			<td style="width:354px;">Axiovert 200</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine 100mg/ml</td>
			<td>Biowet Pulawy</td>
			<td>N/A</td>
			<td>50 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Liquid Paraffin</td>
			<td>Merck Millipore</td>
			<td>8042-47-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">M16 medium EmbryoMax</td>
			<td>Sigma</td>
			<td>MR-016-D</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">M2 medium</td>
			<td>Sigma</td>
			<td>M7167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Magnesium Chloride 1M</td>
			<td style="width:186px;">Sigma Aldrich</td>
			<td style="width:354px;">63069-100ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Microforge</td>
			<td style="width:186px;">Narishige</td>
			<td style="width:354px;">MF-900</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mineral Oil</td>
			<td>Sigma</td>
			<td>M8410-500ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaCl 0.9%</td>
			<td>POLPHARMA OTC</td>
			<td>N/A</td>
			<td>sterile, 5ml ampules</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Operation microscope&#160;</td>
			<td style="width:186px;">Inami Ophthalmic Instruments</td>
			<td style="width:354px;">Deca-21</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Packaging system coding plasmid for lentiviral vectors&#160; (delta R8.2 plasmid)</td>
			<td style="width:186px;">ADDGENE</td>
			<td style="width:354px;">12263</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">PEI reagent (Polyethylenimine, Mw ~ 25,000,),</td>
			<td style="width:186px;">Polysciences, Inc</td>
			<td style="width:354px;">23966-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Penicilin-streptomycin</td>
			<td style="width:186px;">Sigma Aldrich</td>
			<td style="width:354px;">P0781-100ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Phosphate Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture</td>
			<td style="width:186px;">Sigma Aldrich</td>
			<td style="width:354px;">806552-500ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Puller&#160;</td>
			<td style="width:186px;">Sutter Instrument Co.</td>
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			<td style="width:354px;">N/A</td>
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			<td style="width:354px;">N/A</td>
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			<td>470018-868</td>
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generation of transgenic rats using a lentiviral vector approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

Using the protocol described herein, lentiviral vectors that carried the Syn-TDP-43-eGFP construct were produced (physical LV titer = 3.4 x 108/&#181;L) and then could be used for one-cell-stage embryo subzonal injections. Only embryos with two visible pronuclei were subjected to the procedure. The number of injections of viral suspensions was determined experimentally. High implantation efficiency and a simultaneous lack of transgenic offspring were considered indicators of an insufficient number of viral par...

Watch this Scientific Journal Video about Generation of Transgenic Rats using a Lentiviral Vector Approach at JoVE.com

Generation of Transgenic Rats using a Lentiviral Vector Approach

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Transgenic animals are fundamental to modern biomedical research as they allow studies of gene function in living organisms. Various methods of genetic modifications have been applied in animal studies. Here, we present a widely accessible and effective technique that uses lentiviral vectors as a tool for transgene incorporation into the genome of a rat embryo.After human chorionic gonadotropin administration, mate five-week-old Wistar female rats one-to-one with sexually fertile three to 10-month-old Wistar males. At eight to 10 a.m. the next morning, check the females for the presence of a vaginal plug and harvest the oviducts from the females with a mating plug by no later than 10 a.m.into a collection dish containing pre-warmed M2 medium. When all of the oviducts have been collected, transfer the tissues into a 35 millimeter dish containing pre-warmed M2 medium supplemented with 0.5 milligrams per milliliter of hyaluronidase from bovine testes and place the dish under a stereo microscope. Use fine forceps to open the walls of the oviduct and press the ampulla until the embryos are freed.To facilitate the release of the embryos from the cumulus cells, use a glass transfer pipette connected to a mouth-operated aspirator tube to gently pipette the embryos up and down. When all of the embryos have been released, wash the cells a few times in fresh M2 medium to remove the hyaluronidase and cellular debris. Transfer the embryos into individual 50 microliter drops of pre-equilibrated M16 medium covered by mineral oil in a 60 millimeter dish.Then place the dish into a humidified 37 degree Celsius incubator with a 5%carbon dioxide atmosphere until their injection. For lentiviral vector microinjection under the zona pellucida of the one-cell stage embryos, use a pipette puller to prepare microinjection borosilicate glass capillaries with a filament. After every time the filament is changed or a new glass capillary is used, run a Ramp test to set the optimal parameters.Next, use a microloader tip to load approximately two microliters of freshly thawed lentiviral solution per microinjection pipette under a biosafety laminar flow hood. Add a 100 microliter drop of M2 medium to the center of the lid from a 60 millimeter Petri dish. Cover the medium with mineral oil and mount the holding pipette and the virus loaded microinjection capillary onto a micromanipulator.Place the microinjection dish under an inverted microscope and transfer 15 to 20 one-cell stage embryos into the M2 drop in the microinjection dish. Capture one embryo with the holding pipette and use the 400 times magnification and the glass capillary to inject the lentiviral solution under the zona pellucida into the perivitelline space holding the capillary under the zona pellucida for a moment after the virus has been delivered. When all of the embryos have been injected, use a fine pipette to return the injected cells to the culture dish and return the dish to the cell culture incubator until their implantation.To prepare the foster mothers for the embryo transfer, mate sexually mature Sprague-Dawley females with vasectomized males and check the females the next morning for a vaginal plug. After confirming a lack of response to pedal reflex in the anesthetized females with a viable plug, apply ointment to the animal's eyes and shave the fur from the back of each animal. Place the first rat in the prone position on a clean surface on a heating pad under a surgical microscope.Cover the rat with a sterile drape with a small hole cut over the lower back and make an approximately two centimeter skin incision parallel to the lumbar vertebral column. Use sharp scissors to make a cut in the abdominal wall and use forceps to grasp an ovarian fat pad. Pull the ovary and oviduct out of the abdominal cavity onto a piece of 0.9%sodium chloride soaked gauze.Load M2 medium, three bubbles of air, and the embryos into a transfer capillary. Use microscissors to make a small incision into the oviduct between the infundibulum and ampulla and insert the tip of the transfer pipette into the incision. Gently expel the embryos and air bubbles into the oviduct, then use blunt forceps to place the reproductive tract back into the abdominal cavity.Suture the abdominal wall with polyglycolic acid absorbable sutures and close the skin incision with wound clips and place the animal in a clean cage on a warming plate with monitoring until full recumbency. To vasectomize potential mates, after preparing the male rat for surgery as demonstrated, use 70%ethanol and a surgical scrub to disinfect the skin over the testes. Gently press the abdomen to expose the testes in the scrotal sac and cover the rat with a sterile drape with a small hole over the surgical site and use scrotal scissors to make a 0.5 centimeter incision in the middle of the sac.Carefully push the testis to the left and locate the vas deferens between the testis and midline as a white duct with a single blood vessel. Use watchmaker's forceps to gently pull the vas deferens out of the scrotal sac and holding the vessel with forceps, use fine scissors to remove a one centimeter fragment from the duct. Repeat the procedure for the second testis as just demonstrated and close the skin with polyglycolic acid absorbable sutures.Then place the rat into a clean cage on a warming plate with monitoring until full recumbency. In this representative experiment in which single embryos were injected multiple times, eight rats were born from embryos that were injected two times, three of which were confirmed to carry the transgene. One of the founders did not transfer the transgene to offspring and three foster females were used for each experimental setup.The chosen approach allowed the generation of stable transgenic rat lines that expressed the TDP-43-eGFP fusion protein under the control of the neuronal synapse in one promoter throughout the central nervous system. Lentivirus-based transgenesis resulted in a single copy insertion of the transgene as demonstrated by quantitative PCR. When this method was used for other lentiviral vectors, an approximately 90%survival rate was observed.The percentage of embryos that survived the pronuclear injections was significantly lower. Overall, these results suggest that pregnant female rats can be used as foster mothers with a comparable efficiency. Notably, the numerical data that were analyzed for individual rounds of microinjection indicated that the effectiveness of implantation depended directly on the number of injections into one embryo and indirectly on the viral load.The use of lentiviral vectors guarantees the genomic integration and transmission of the transgene and facilitates a high survival rate of the micromanipulated embryos even when multiple subdermal injections are performed. This procedure may be effectively applied to other species and can be considered an alternative for conventional transgenesis.

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