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 production
<ol>
	<li>Transfection of HEK 293T cells 
	NOTE: The protocol that is presented herein is designed for the transfection of twenty &#216;10 cm culture dishes that produces approximately 200 mL of crude vector supernatant.
	<ol>
		<li>Culture HEK 293T cells in DMEM medium that is supplemented with fetal bovine serum (10%, v/v) in a humidified CO2 incubator at 37 &#176;C. For transfection, prepare twenty 10 cm diameter plates, and seed 1.5&#8211;2 x 106 HEK 293T cells per dish.</li>
		<li>When confluence reaches ~70%, transfect the cells using polyethylenimine (PEI) reagent, pH 7.0, at a ratio of 3 &#956;g of PEI per 1 &#956;g of DNA.
		<ol>
			<li>Prepare the transfection mixture for five plates (prepare the number of repetitions according to the total number of dishes). To 1 mL of Dulbecco&#8217;s Modified Eagle Medium (DMEM; without serum), add the mixture of three plasmids so they reach a final amount of 25 &#181;g of VSVg plasmid, 50 &#181;g of delta R8.2, and 50 &#181;g of coding plasmid.</li>
			<li>Pipette up and down, and add 125 &#181;L of PEI at a concentration of 3 &#181;g/&#181;L. Incubate at room temperature for 15 min, inverting the tube three times during incubation. Add 200 &#181;L of the transfection mixture per plate. Next, incubate the plates in a humidified CO2 incubator at 37 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Concentration of lentiviral vectors
	<ol>
		<li>Forty-eight hours after transfection, harvest the medium that contains LV particles. Use 50 mL conical tubes. 
		NOTE: When using a plasmid with a fluorescent tag, cells can be visualized at this point to verify transfection efficiency. A new portion of DMEM medium can be added, and cells may be incubated for an additional 24 h. The LV yield is comparable when collected at the 48 and 72 h time points after transfection.</li>
		<li>Centrifuge the medium at 3,000 x g for 5 min and room temperature to remove detached cells.</li>
		<li>Filter the supernatant (0.45 &#956;m) and pour it into new tubes. 
		NOTE: This step can be omitted.</li>
		<li>Add DNase I (RNase-free, 1 &#956;g/mL) and MgCl2 (1 mM), and incubate in a water bath at 37 &#176;C for 15 min.</li>
		<li>Transfer the medium to disposable polyethylene tubes, and ultracentrifuge in a swinging rotor at 115,000 x g and 4 &#176;C for 1.5 h.</li>
		<li>After centrifuging, gently drain the walls of the tubes from the medium residues.</li>
		<li>Soak the pellet with sterile phosphate-buffered saline (PBS; 70&#8211;80 &#181;L per tube).</li>
		<li>Incubate for 2 h at 4&#8211;8 &#176;C.</li>
		<li>Resuspend the viral vectors in PBS by gentle pipetting. 
		CAUTION: Avoid foaming.</li>
		<li>Transfer to a 1.5 mL centrifuge tube and centrifuge at 7,000 x g and 4 &#176;C for 30 s. Transfer the supernatant to a new tube. Repeat this step until no cellular debris pellet is visible.</li>
		<li>Aliquot and freeze at -80 &#176;C. Avoid refreezing the LV aliquot.</li>
	</ol>
	</li>
	<li>Determination of virus titer using quantitative polymerase chain reaction 
	NOTE: The titration of viral vectors is performed using quantitative PCR (qPCR). This method is based on amplifying a double-stranded 84 bp long DNA fragment within the long terminal repeat region of the viral genome15.
	<ol>
		<li>Prepare the standard curve by making serial dilutions of the LV-coding plasmid: 1:500, 1:1,000, 1:5,000, 1:10,000, 1:100,000, and 1:1,000,000. Determine the number of copies of the plasmid that is used for the standard curve. Use the following formula: number of copies/&#181;L = (concentration [g/&#181;L] x 6.02 x 1023 [number/mol]) / (660 [g/mol] x plasmid size [bp]), where 6.02 x 1023 number/mol is Avogadro&#8217;s number, and 660 g/mol is the bp weight. 
		NOTE: Online copy number calculators may be used.</li>
		<li>Prepare dilutions of the lentiviral suspension: 1:100, 1:500, and 1:1,000.</li>
		<li>Prepare the reaction mixture (volumes per well): 10 &#181;L of qPCR Mastermix, 1 &#181;L of 10 &#956;M Forward primer, 1 &#181;L of 10 &#956;M reverse primer, and 7 &#181;L of H2O. Pipette the mixture into the wells of 96-well plates. 
		NOTE: Forward primer: 5&#8217;-AGCTTGCCTTGAGTGCTTCA. Reverse primer: 5&#8217;-TGACTAAAAGGGTCTGAGGGA.</li>
		<li>Add 1 &#181;L of each standard dilution and lentiviral suspension in triplicate.</li>
		<li>Run the qPCR according to the following parameters: 50 &#176;C for 2 min, 96 &#176;C for 5 min, and 35 cycles of 96 &#176;C for 20 s, 60 &#176;C for 40 s, and 70 &#176;C for 1 min, followed by melt curve stage: 95 &#176;C for 1 min and 60 &#176;C at 30 s.</li>
		<li>Analyze the results by comparing the number of molecules that are received for each dilution to the standard curve. Determine the concentration of vector molecules as the average of three replicates for each dilution. 
		NOTE: The presented quantification gives the physical concentration of the viral particles. It should not be treated as a functional titer.</li>
	</ol>
	</li>
</ol>
2. Generation of transgenic rats
<ol>
	<li>Superovulation and collection of fertilized embryos
	<ol>
		<li>Administer gonadotropins. 
		NOTE: To increase the number of collected embryos (approximately 30 per female), use immature 5-week-old Wistar females for hormonal stimulation.
		<ol>
			<li>On Day 1 (12 PM&#8211;1 PM), intraperitoneally inject pregnant mare&#8217;s serum gonadotropin (PMSG; 25 IU per female). Prepare 1 mL aliquots of working solution at a concentration of 125 IU/mL by dissolving hormone powder in 0.9% NaCl. Store at -20 &#176;C for up to 1 month or -80 &#176;C for up to 6 months.</li>
			<li>On day 3 (12 PM&#8211;1 PM), intraperitoneally inject human chorionic gonadotrophin (hCG; 30 IU per female). Prepare 1 mL aliquots of working solution (150 IU/mL) by dissolving hormone powder in 0.9% NaCl. Store at -20 &#176;C for up to 1 month or -80 &#176;C for up to 6 months.</li>
		</ol>
		</li>
		<li>After hCG administration, mate females 1:1 with sexually fertile males (3-10 months old).</li>
		<li>The next morning (day 4 at 8&#8211;10 AM), check the females for the presence of a vaginal plug. Check the vaginal opening for the presence of a whitish mating plug, which for best visualization should be checked early in the morning after the mating night. For embryo collection, use only females with a visible plug.</li>
		<li>Collect embryos at 10 AM. Sacrifice the animals to excise the oviducts, and collect the oviducts in a dish with pre-warmed M2 medium.
		<ol>
			<li>Transfer the oviducts to a 35 mm dish that contains pre-warmed M2 medium with hyaluronidase from bovine testes at a concentration of 0.5 mg/mL.</li>
			<li>Open the walls of the oviduct using fine forceps under a stereomicroscope and press the ampulla (i.e., the swollen part of the oviduct that contains fertilized embryos that are surrounded by cumulus cells) until the embryos are freed. 
			NOTE: Hyaluronidase enzymatically digests cumulus cells, releasing embryos. 
			CAUTION: Prolonged exposure to hyaluronidase is deleterious to embryos; therefore, this step should last no longer than 5 min.</li>
			<li>To facilitate the release of embryos from cumulus cells, gently pipette them up and down using a glass transfer pipette that is connected to a mouth-operated aspirator tube.
			<ol>
				<li>To produce the transfer pipette, pull a glass Pasteur pipette over a flame to produce a straight ~5-10 cm tip. Break the pipette leaving a ~4 cm tip.</li>
			</ol>
			</li>
			<li>Wash the embryos a few times in M2 medium to remove hyaluronidase and cellular debris. Transfer the embryos to a 60 mm dish that contains (~50 &#181;L) drops of pre-equilibrated M16 medium, covered by liquid paraffin or mineral oil, in a humidified 37 &#176;C incubator with a 5% CO2 atmosphere.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Microinjection of lentiviral vectors to one-cell-stage embryo under the zona pellucida 
	NOTE: Use one-cell-stage embryos with two visible pronuclei for microinjection (Figure 1).
	<ol>
		<li>Thaw the LV aliquot at room temperature and centrifuge at 10,000 x g and RT for 2 min to pellet any remaining cellular debris.</li>
		<li>Microinjection setup
		<ol>
			<li>Prepare glass holding pipettes (borosilicate glass capillary) using a microforge. Pull the glass capillary over a flame to produce a 5&#8211;10 cm tip. Break the pipette leaving a ~4 cm tip. The outside diameter should be ~80&#8211;120 &#181;m. 
			NOTE: Ensure that the pipette tip is perfectly straight and smooth.</li>
			<li>Assemble the pulled pipette in a microforge with the tip in front of the heating filament. Heat the filament very close to the pipette tip and allow it to shrink to a diameter of ~15 &#181;m (approximately 20% of embryo size). Position the pipette perpendicularly to the heating filament, 2&#8211;3 mm from the pipette tip, and begin to heat. The glass will soften. Heat until it reaches a 15&#176; angle.</li>
			<li>Prepare microinjection borosilicate glass capillaries with a filament using a pipette puller. Insert the capillary in the pulling chamber. Run a ramp test (for the first time for new glass and every time after changing the filament). Set the Heat to the ramp value -10, Pull to 100, Velocity to 150 and Time to 100. 
			NOTE: Modify the parameters to obtain an optimal injection capillary.</li>
			<li>Under a biosafety laminar flow hood, load approximately 2 &#181;L of the viral solution into the microinjection pipette with a microloader tip.</li>
			<li>Prepare a microinjection dish (lid of 60 mm Petri dish) with a 100 &#181;L drop of M2 medium (in the middle), covered by liquid paraffin or mineral oil.</li>
			<li>Mount the holding pipette and microinjection capillary that is loaded with viral solution to a micromanipulator and microinjection dish under an inverted microscope.</li>
		</ol>
		</li>
		<li>Perform the microinjection.
		<ol>
			<li>Transfer 15&#8211;20 one-cell-stage embryos to the M2 drop on the microinjection dish. Hold the embryo using a holding pipette.</li>
			<li>Using 400x magnification, inject the LV solution under the zona pellucida to the perivitelline space using the glass capillary that is connected to an automatic injector. Hold the capillary under the zona pellucida for a moment. 
			NOTE: Using gentle positive pressure, the viral solution will flow continuously out of the injection capillary, but the volume of the suspension that is delivered cannot be controlled.</li>
			<li>Using a fine pipette, return the embryos to the culture dish in the incubator at 37 &#176;C in a 5% CO2 atmosphere. The number of injections of one zygote may vary and can be adapted based on the viral vector concentration. 
			NOTE: The injected embryos can be transferred to foster mothers at the one-cell stage or incubated O/N in M16 medium before being transferred at the two-cell stage. Prolonged in vitro culture of rat embryos should be avoided.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfer of injected embryos to foster mothers
	<ol>
		<li>Prepare foster mothers by mating sexually mature SD females with fertile BN males or with vasectomized SD males (the vasectomy procedure is described in section 3 below) on day 3 (for transferring embryos at the one-cell stage) or day 4 (for transferring embryos at the two-cell stage). 
		NOTE: For oviduct transfer, use 0.5 days post coitum (dpc) females.</li>
		<li>The next morning, check SD females for a vaginal plug, and use only those with a visible plug.</li>
		<li>Perform embryo transfer. 
		NOTE: Conduct the surgical procedure with sterile instruments under a stereomicroscope. Before the day of surgery, autoclave scissors, fine forceps, needle holder, and scalpel holder.
		<ol>
			<li>Anesthetize a female with i.p. administration of ketamine (50 mg/kg) and medetomidine (0.5 mg/kg) solution.Test for reflexes to confirm anesthesia before starting the surgical procedure.</li>
			<li>Inject the animal subcutaneously with tolefenamic acid (2 mg/kg), butorphanol tartrate (1 mg/kg), and enrofloksacin (5&#8211;10 mg/kg) to prevent inflammation, pain, and infection, respectively.</li>
			<li>Apply ophthalmic ointment lubrication to both eyes to prevent corneal drying. Shave the fur from the back, and sterilize the skin with surgical scrub followed by 70% alcohol using sterile non-adhering pads. Allow the skin to dry.</li>
			<li>Inject the animal subcutaneously with 100 &#181;L of 0.25% bupivacaine (local anesthetic) at the incision site. Transfer the animal in a prone position to a clean surface on a heating pad under the objective of a surgical microscope. Cover the rat with a sterile drape with a small hole cut over the lower back.</li>
			<li>Perform an approximately 2 cm skin incision, parallel to the lumbar vertebral column.</li>
			<li>Using sharp scissors, make a cut in the abdominal wall. Grab an ovarian fat pad using forceps, and pull out the ovary and oviduct and place them on gauze that is wetted with 0.9% NaCl.</li>
			<li>Aspirate M2 medium, three bubbles of air, and the embryos into the transfer capillary. Recommended total number of embryos to be transferred (unilateral or bilateral): pregnant female (&#8804; 15&#8211;16 embryos), pseudopregnant female (&#8804; 30 embryos).</li>
			<li>Make a small incision in the oviduct (between the infundibulum and ampulla) using micro-scissors, and insert the transfer pipette in the oviduct.</li>
			<li>Gently expel embryos and air bubbles from the pipette to the oviduct. With blunt forceps, place the reproductive tract back in the abdominal cavity.</li>
			<li>Suture the abdominal wall with polyglycolic acid absorbable sutures and close the skin incision with wound clips. Depending on the number of embryos that are available, repeat this procedure for the other oviduct.</li>
			<li>Inject the animal intraperitoneally with atipamezole (0.5 mg/kg) to reverse the effect of anesthesia.</li>
			<li>Transfer the animal to a clean cage and keep it on a warming plate to fully recover from anesthesia. Delivery in rats occurs after ~21 days. 
			NOTE: When male BN rats are used for mating, only white pups are potentially transgenic; brown pups are from natural pregnancy.</li>
			<li>Collect tissue fragments (preferably from the ear) to genotype 3-week-old pups.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Vasectomy
NOTE: Before the day of surgery, autoclave scissors, fine forceps and needle holder.
<ol>
	<li>Anesthetize a 5-week-old male SD rat with i.p. administration of ketamine (50 mg/kg) and medetomidine (0.5 mg/kg) solution.&#160;Test for reflexes to confirm anesthesia before starting the surgical procedure.</li>
	<li>Administer tolfenamic acid (2 mg/kg), butorphanol tartrate (1 mg/kg), and enrofloksacin (5&#8211;10 mg/kg) subcutaneously to prevent inflammation, pain, and infection, respectively.</li>
	<li>Apply ophthalmic ointment lubrication to both eyes to prevent corneal drying. Place the rat supine on a clean surface on a heating pad, and sterilize the skin on the testes with surgical scrub followed by 70% alcohol using sterile non-adhering pads. Allow the skin to dry. Cover the rat with a sterile drape with a small hole cut over the testes. Gently press the abdomen to expose the testes in the scrotal sac.</li>
	<li>Using surgical scissors, make a ~0.5 cm incision in the middle of the scrotal sac. Locate the midline wall (whitish line) between the testes.</li>
	<li>Make a 5 mm incision in the testis membrane close to the left side of the midline wall.</li>
	<li>Carefully push the testis to the left and locate vas deferens (between the testis and midline) as a white duct with a single blood vessel.</li>
	<li>Gently pull the vas deferens out of the scrotal sac using a watchmaker&#8217;s forceps. Hold the vas deferens with one pair of forceps, and cut it with fine scissors (or cauterize with red-hot tips of a second pair of forceps). Remove a ~1 cm fragment of the duct. 
	NOTE: If cauterization is performed, hold the tip of the second pair of forceps in the flame.</li>
	<li>Repeat the above procedure for the other testis. Suture the skin with polyglycolic acid absorbable sutures and inject the animal intraperitoneally with atipamezole (0.5 mg/kg).</li>
	<li>Place the rat in a clean cage on a warming plate until the animal recovers from anesthesia. 
	NOTE: Males can be used in the test matings after a ~2-week recovery period. After sterility is confirmed, they can be used for pseudopregnancy induction.</li>
</ol>

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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=Optimization+of+embryo+transfer+protocols+for+mice.">Optimization of embryo transfer protocols for mice.</a> Theriogenology. 46 (7), 1267-1276 (1996).</li><li>van den Brandt, J., Wang, D., Kwon, S. H., Heinkelein, M., Reichardt, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirally+generated+eGFP-transgenic+rats+allow+efficient+cell+tracking+in+vivo.">Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo.</a> Genesis. 39 (2), 94-99 (2004).</li><li>Remy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Lentiviral+Vectors+to+Obtain+Transgenic+Rats.">The Use of Lentiviral Vectors to Obtain Transgenic Rats.</a> Rat Genomics: Methods and Protocols. 597, 109-125 (2010).</li></ol>

The author (W.K.) has rights to the patent, &#8220;Method of producing of a transgenic animal,&#8221; from the patent office of the Republic of Poland (no. P 355353; 21.03.2008).

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.

<table><tbody><tr><td>7500 Real Time PCR System</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Aerrane (isoflurane)</td><td>Baxter</td><td>FDG9623</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma</td><td>A5177-5EA</td><td></td></tr><tr><td>Atipam 5 mg/ml</td><td>Eurovet Animal Health BV</td><td>N/A</td><td>0.5 mg/kg</td></tr><tr><td>Baytril 25 mg/ml (enrofloksacin)</td><td>Bayer</td><td>N/A</td><td>5-10 mg/kg</td></tr><tr><td>Borosilicate glass capillaries with filament GC100TF-15</td><td>Harvard Apparatus Limited</td><td>30-0039</td><td>injection capillary</td></tr><tr><td>Bupivacaine 25 mg/ml</td><td>Advanz Pharma</td><td>N/A</td><td>0.25% in&amp;nbsp; 0.9% NaCl</td></tr><tr><td>Butomidor 10 mg/ml (butorphanol&amp;nbsp; tartrate)</td><td>Orion Pharma</td><td>N/A</td><td>1 mg/kg</td></tr><tr><td>CELLSTAR Tissue Cell Culture Dish 35-mm</td><td>Greiner Bio-One</td><td>627160</td><td></td></tr><tr><td>CELLSTAR Tissue Cell Culture Dish 60-mm</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>CellTram Oil</td><td>Eppendorf</td><td>5176 000.025</td><td></td></tr><tr><td>Cepetor (Medetomidine) 1 mg/ml</td><td>cp-pharma</td><td>N/A</td><td>0.5 mg/kg</td></tr><tr><td>Chorulon, Human Chorionic Gonadotrophin&amp;nbsp;</td><td>Intervet</td><td>N/A</td><td>150 IU/ ml ml 0.9% NaCl</td></tr><tr><td>DMEM low glucose</td><td>Sigma Aldrich</td><td>D6048</td><td></td></tr><tr><td>DNase, RNase-free</td><td>A&amp;amp;A Biotechnology</td><td>1009-100</td><td></td></tr><tr><td>EmbryoMax Filtered Light Mineral Oil</td><td>Sigma</td><td>ES-005-C</td><td></td></tr><tr><td>Envelope protein coding plasmid for lentiviral vectors (VSVg plasmid)&amp;nbsp;</td><td>ADDGENE</td><td>14888</td><td></td></tr><tr><td>FemtoJet</td><td>Eppendorf</td><td>4i /5252 000.013</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>F9665-500ML</td><td></td></tr><tr><td>Folligon, Pregnant Mare&amp;rsquo;s Serum Gonadotropin&amp;nbsp;</td><td>Intervet</td><td>N/A</td><td>125 IU/ml in .9% NaCl</td></tr><tr><td>HEK 293T cells&amp;nbsp;</td><td>ATCC</td><td>ATCC CRL-3216</td><td></td></tr><tr><td>Hyaluronidase from Bovine Testis&amp;nbsp;</td><td>Sigma</td><td>H4272-30MG</td><td>0.5 mg/ml in M2 medium</td></tr><tr><td>Inverted Microscope&amp;nbsp;</td><td>Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Ketamine 100mg/ml</td><td>Biowet Pulawy</td><td>N/A</td><td>50 mg/kg</td></tr><tr><td>Liquid Paraffin</td><td>Merck Millipore</td><td>8042-47-5</td><td></td></tr><tr><td>M16 medium EmbryoMax</td><td>Sigma</td><td>MR-016-D</td><td></td></tr><tr><td>M2 medium</td><td>Sigma</td><td>M7167</td><td></td></tr><tr><td>Magnesium Chloride 1M</td><td>Sigma Aldrich</td><td>63069-100ML</td><td></td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-900</td><td></td></tr><tr><td>Mineral Oil</td><td>Sigma</td><td>M8410-500ML</td><td></td></tr><tr><td>NaCl 0.9%</td><td>POLPHARMA OTC</td><td>N/A</td><td>sterile, 5ml ampules</td></tr><tr><td>Operation microscope&amp;nbsp;</td><td>Inami Ophthalmic Instruments</td><td>Deca-21</td><td></td></tr><tr><td>Packaging system coding plasmid for lentiviral vectors&amp;nbsp; (delta R8.2 plasmid)</td><td>ADDGENE</td><td>12263</td><td></td></tr><tr><td>PEI reagent (Polyethylenimine, Mw ~ 25,000,),</td><td>Polysciences, Inc</td><td>23966-1</td><td></td></tr><tr><td>Penicilin-streptomycin</td><td>Sigma Aldrich</td><td>P0781-100ML</td><td></td></tr><tr><td>Phosphate Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma Aldrich</td><td>806552-500ML</td><td></td></tr><tr><td>Puller&amp;nbsp;</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Reflex Clip Applier/Reflex Clips</td><td>World Precision Instruments</td><td>500345/500346</td><td></td></tr><tr><td>Safil, polyglycolic acid, braided, coated, absorbable threads</td><td>B.Braun Surgical</td><td>1048029</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Surgical Sewing Thread</td><td>B.Braun&amp;nbsp;</td><td>C1048040</td><td></td></tr><tr><td>SYBR Green PCR Master Mix</td><td>Applied Biosystem</td><td>4334973</td><td></td></tr><tr><td>Tolfedine 4% (tolfenamic acid)</td><td>Vetoquinol</td><td>N/A</td><td>2 mg/kg</td></tr><tr><td>TransferMan NK2</td><td>Eppendorf</td><td>N/A</td><td></td></tr><tr><td>Trypsin EDTA solution</td><td>Sigma Aldrich</td><td>T3924-500ML</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-100 XP&amp;nbsp;</td><td></td></tr><tr><td>VacuTip</td><td>Eppendorf</td><td>5175108.000</td><td>holders capillary</td></tr><tr><td>Vita-POS</td><td>Ursapharm</td><td>N/A</td><td>eye ointment</td></tr><tr><td>Warming Plate</td><td>Semic</td><td>N/A</td><td></td></tr><tr><td>Watchmaker Forceps</td><td>VWR</td><td>470018-868</td><td></td></tr></tbody></table>

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 ...

generation-of-transgenic-rats-using-a-lentiviral-vector-approach

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.5K Views.  Polish Academy of Sciences. 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.

Video: Generation of Transgenic Rats using a Lentiviral Vector Approach

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Immunology and Infection

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Genetics

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Neuroscience

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Medicine

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

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

Biology

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

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Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk