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 border="0" cellpadding="0" cellspacing="0" style="width:1208px;" width="1209">
	<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>
			<td style="width:354px;">P-97</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Reflex Clip Applier/Reflex Clips</td>
			<td style="width:186px;">World Precision Instruments</td>
			<td style="width:354px;">500345/500346</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Safil, polyglycolic acid, braided, coated, absorbable threads</td>
			<td style="width:186px;">B.Braun Surgical</td>
			<td style="width:354px;">1048029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">Stereomicroscope</td>
			<td style="width:186px;">Olympus</td>
			<td style="width:354px;">SZX16</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical Sewing Thread</td>
			<td>B.Braun&#160;</td>
			<td>C1048040</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">SYBR Green PCR Master Mix</td>
			<td style="width:186px;">Applied Biosystem</td>
			<td style="width:354px;">4334973</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tolfedine 4% (tolfenamic acid)</td>
			<td>Vetoquinol</td>
			<td>N/A</td>
			<td>2 mg/kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:496px;">TransferMan NK2</td>
			<td style="width:186px;">Eppendorf</td>
			<td style="width:354px;">N/A</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:496px;">Trypsin EDTA solution</td>
			<td style="width:186px;">Sigma Aldrich</td>
			<td style="width:354px;">T3924-500ML</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:496px;">Ultracentrifuge</td>
			<td style="width:186px;">Beckman Coulter</td>
			<td style="width:354px;">Optima L-100 XP&#160;</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:496px;">VacuTip</td>
			<td style="width:186px;">Eppendorf</td>
			<td style="width:354px;">5175108.000</td>
			<td style="width:172px;">holders capillary</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vita-POS</td>
			<td>Ursapharm</td>
			<td>N/A</td>
			<td>eye ointment</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:496px;">Warming Plate</td>
			<td style="width:186px;">Semic</td>
			<td style="width:354px;">N/A</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Watchmaker Forceps</td>
			<td style="width:186px;">VWR</td>
			<td>470018-868</td>
			<td style="width:172px;"></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

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

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

In Silico Modeling Method for Computational Aquatic Toxicology of Endocrine Disruptors: A Software-Based Approach Using QSAR Toolbox

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological systems. In particular, quantitative structure-activity relationship (QSAR) models have been increasingly applied to assess the environmental effects of a plethora of toxic materials. In recent years, some more highlighted types of toxicants are endocrine disruptors (EDs, which are chemicals that can interfere with any hormone-related metabolism). Because EDs may significantly affect animal development and reproduction, rapidly predicting the adverse effects of EDs using in silico techniques is required. This study presents an in silico method to generate prediction data on the effects of representative EDs in aquatic vertebrates, particularly fish species. The protocol describes an example utilizing the automated workflow of the QSAR Toolbox software developed by the Organization for Economic Co-operation and Development (OECD) to enable acute ecotoxicity predictions of EDs. As a result, the following are determined: (1) calculation of the numerical correlations between the concentration for 50% of lethality (LC50) and octanol-water partition coefficient (Kow), (2) output performances in which the LC50 values determined in experiments are compared to those generated by computations, and (3) the dependence of estrogen receptor binding affinity on the relationship between Kow and LC50.

Quantitative structure-activity relationship (QSAR) modeling is a representative bioinformatics-assisted method in toxicological screening. This protocol demonstrates how to computationally assess the risks of endocrine disruptors (EDs) in aquatic environments. Utilizing the OECD QSAR Toolbox, the protocol implements an in silico assay for analyzing toxicity of EDs in fish.

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...