We thank Xia Liu (Regeneron Pharmaceuticals) for initially teaching us the HGD technique and Dr. Russell Thomson (Albert Einstein College of Medicine) for his continual guidance in various aspects of the technology. This work was funded by Hunter College, CUNY start up funds and NSF Bread award IOS-1249166. We thank the NYULMC Histopathology Core NYUCI Center Support Grant, NIH/NCI 5 P30CA16087-31 for histology on mouse livers.

All experiments described here were approved by the Institutional Animal Care and Use Committee of Hunter College, City University of New York.
1.&#160;Preparation of endotoxin-free plasmid DNA
<ol>
	<li>Pick a single colony of bacteria containing the gene of interest in a mammalian expression vector from a freshly streaked selective plate.</li>
	<li>Follow the recommendations found in the handbook of a commercially available endotoxin-free plasmid purification kit for growing and harvesting bacteria.</li>
	<li>Purify the endotoxin-free plasmid DNA from bacterial cells by following the protocol of a commercially available endotoxin-free plasmid purification kit.</li>
	<li>Use endotoxin-free plastic ware and handle DNA with care to ensure that endotoxin is not re-introduced into the DNA sample after the removal step.</li>
	<li>Measure the concentration of endotoxin-free plasmid DNA by determining its absorbance at 260 nm using a micro-volume UV spectrophotometer as described below or a standard, cuvette-based spectrophotometer.
	<ol>
		<li>Clean the lower and upper optical surfaces of the micro spectrophotometer sample retention system as follows. Pipette 1 &#956;l of clean deionized water onto the lower optical system. Close the lever arm and tap it a few times to bathe the upper optical system, then open lever and wipe clean with a tissue.</li>
		<li>Open the software of the micro spectrophotometer and select the nucleic acids module.</li>
		<li>Place 1 &#956;l clean deionized water on the lower optical system, lower the lever arm and select &#8220;initialize&#8221; from the program software. Once initialization is complete, clean both optical surfaces with a tissue..</li>
		<li>Perform blank measurement by loading 1 &#956;l of endotoxin-free TE buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA) and selecting &#8220;blank&#8221; from the program software. Once blank measurement is done, clean both optical surfaces with a tissue.</li>
		<li>Perform sample measurement by loading 1 &#956;l of endotoxin-free DNA sample and selecting &#8220;measure&#8221; from the program software. Record the DNA concentration. Assess DNA purity by recording the absorbance ratio 260/280 nm that appears on the screen. Since the use of pure DNA (260/280 ratio of 1.8) for HGD is highly desirable, avoid using a DNA sample with a 260/280 ratio below 1.7. Once measurement is done, clean both optical surfaces with a tissue.</li>
	</ol>
	</li>
</ol>
2.&#160;Weighing of mice
<ol>
	<li>Mark mice in a manner appropriate for the strain. Note: marking of the tail is not recommended for this procedure.
	<ol>
		<li>Mark mouse species that have white fur (e.g. Swiss Webster) on their backs with a black marker. Reapply marks over time as necessary.</li>
		<li>Mark mouse species that have black fur (e.g. C57BL/6) by ear punching several days in advance.</li>
	</ol>
	</li>
	<li>Weigh mice individually by gently placing them in an animal weighing pan or bucket placed on a digital laboratory balance. Record the weight of each mouse used in the experiment, on the day of the experiment.</li>
</ol>
3.&#160;Preparation of syringes
<ol>
	<li>Preparation of the injection mix
	<ol>
		<li>Calculate the amount of DNA needed by assuming 5 - 50 &#956;g endotoxin-free plasmid DNA for the injection of each mouse.
		<ol>
			<li>Determine the optimal amount of plasmid DNA experimentally.</li>
			<li>When determining the amount of DNA required, assume injection of one additional mouse per group, as proper loading of the syringes will require some extra injection mix. To help calculations, consider the following example: inject 4 experimental and 4 control mice, each weighing 25 g, with 50 &#956;g plasmid DNA per mouse. To determine the amount of DNA needed in this case, calculate with 5 mice per group: 5 x 50 &#956;g = 250 &#956;g DNA needed for each group.</li>
		</ol>
		</li>
		<li>Determine the amount of saline (0.9% sodium chloride) needed by assuming 10% of body weight for the injection of each mouse.
		<ol>
			<li>According to the above example, inject each 25 g mouse with 50 &#956;g of plasmid DNA in 2.5 ml saline (2.5 g liquid).</li>
			<li>When determining the amount of saline required for an entire group of mice, assume injection of an additional mouse per group to allow for proper loading of the syringes. Considering the experiment given, calculate the amount of saline needed for 5 mice in each group: 5 x 2.5 ml =12.5 ml saline for each group (or 25 ml total). Note: If mice within the same group are not the same weight (more than 10% difference in body weight), prepare injection mixes separately.</li>
		</ol>
		</li>
		<li>Use preservative- and endotoxin-free, sterile saline that has been approved for human use, in 10 ml, multiple use vials.</li>
		<li>Using a 20-gauge needle (0.9 mm x 25 mm) attached to a luer-lock tip of a 20 ml syringe, withdraw the liquid from saline vials and empty the syringes into a 50 ml conical tube. To help accurate pipetting of saline during the preparation of the DNA-saline injection mixes, collect more saline than needed for the injection of all the mice in the experiment. For the experiment above, withdraw saline from 3, 10 ml vials (a total of approximately 30 ml) even if the calculated amount of saline needed for the experiment is only 25 ml.</li>
		<li>Pipette the calculated amount of DNA into a different 50 ml conical tube. Take care not to touch the side of the tube with the pipette body to avoid introduction of endotoxin. According to the example, pipette 250 &#956;g of control and 250 &#956;g of experimental plasmid DNA into separate conical tubes.</li>
		<li>Add the required amount of saline to the DNA using a serological pipette. Considering the sample experiment, pipette 12.5 ml saline to each of the master mixes (experimental and control).</li>
		<li>Allow all solutions to reach room temperature prior to injection.</li>
	</ol>
	</li>
	<li>Preparation of syringes
	<ol>
		<li>For each mouse, fill a sterile, 3 ml, luer-lock syringe with the DNA-saline mix using a sterile, 20-gauge needle (0.9 mm x 25 mm). Take care to avoid air bubbles. Avoid using higher volume syringes as the flow rate of injection cannot be controlled very well, and it is difficult to manipulate larger syringes in one hand. Eject any excess DNA-saline mix back into the conical tube as this may be re-used.</li>
		<li>Switch the needle to a sterile, 27-gauge needle. Fill the needle with liquid completely without introducing air bubbles. Adjust the volume of the DNA-saline mix as calculated based on the weight of the mouse.</li>
		<li>Do not allow the uncapped needle to touch any non-sterile surface. If necessary, needles may be re-capped using the one-handed technique: place the cap on a flat surface, insert the needle without holding onto the cap with the other hand and press the capped needle against a firm object to secure the cap onto the needle. The syringes are now ready for tail-vein injections.</li>
	</ol>
	</li>
</ol>
4.&#160;Hydrodynamic DNA delivery
<ol>
	<li>Note that anesthetizing mice may reduce viability. Avoid performing HGD in a room that is too cold, as this will also reduce viability. If the room has an ambient temperature of 20 &#176;C or if using anesthesia, place the mice on a heating pad set at 37 &#176;C post procedure to avoid loss of any animals. If using anesthesia, ensure that the mouth and snout of the mouse are unobstructed while on the heat pad and observe the mouse until it fully recovers. Do not withhold food or water from the mice prior to HGD.</li>
	<li>Warm the mice to dilate blood vessels.
	<ol>
		<li>Place the mouse cage (with bedding included) on a heat pad for 5 min so that the temperature of the bedding is approximately 38-39 &#176;C. The temperature should be low enough to keep the mice on the heat pad for an extended period.</li>
		<li>Alternatively, place mouse into a dorsal access restrainer with minimal restraint. Warm the tail of the mouse for 1 min by gently holding it with a piece of gauze wetted in warm water. Allow mouse to re-position, if necessary. Wipe the tail with a tissue to dry.</li>
		<li>Alternatively, warm mouse by placing the cage under a heat lamp for no more than 2 min. If using a heat lamp, exercise caution to avoid overheating of the mouse.</li>
	</ol>
	</li>
	<li>Large-volume tail vein injection
	<ol>
		<li>Immobilize a dorsal access restrainer on a flat surface by laboratory tape.</li>
		<li>Holding by the tail, place the mouse gently into the restrainer and insert the plug. Use as little restraint as necessary to keep the tail immobilized. Ensure that the mouse is breathing freely.</li>
		<li>If the mouse is in severe distress at any point during the procedure, remove the mouse from the restrainer immediately and allow it to rest.</li>
		<li>Position the mouse so one of the lateral caudal veins is visible. Locate the lateral caudal veins by looking straight down at the back of the mouse: the red line running in the middle of the tail is an artery, while the blue lines on either side of the tail are the lateral caudal veins.</li>
		<li>Wipe the tail of the mouse thoroughly with an alcohol swab.</li>
		<li>Using the non-injecting hand, hold the tail tightly between the index and middle fingers so that the tail passes under the index finger, over the middle and third fingers and under the little finger. Allow the thumb to remain free to move. (Figure 1A)</li>
		<li>Using the other hand, wipe the area to be injected again with an alcohol swab. Allow area to dry.</li>
		<li>Pick up the loaded syringe with the injecting-hand and hold it by the barrel between the thumb and the other four digits. Do not hold onto the plunger.</li>
		<li>Position the syringe parallel to the tail with the needle pointing toward the body of the mouse and the bevel (slanting edge) of the needle facing up.</li>
		<li>Insert the needle into the tail vein. Proceed with the injection only if the vein is correctly located, which is evident from the needle sliding in with no resistance. Note that the depth of needle insertion is a matter of personal preference however, full insertion is not recommended due to an increased risk of shearing of the vein. Avoid moving the needle.</li>
		<li>Using the thumb of the tail-holding hand, close down on the hub of the needle and press hub against the tail to hold needle in position. Do not apply extreme pressure and do not hold onto needle shaft, as these will impede the flow of liquid into the vein. Hold the tail with the index finger loosely at this point so as not impede injection (Figure 1A).</li>
		<li>Re-position the syringe-holding hand so that the index finger and middle finger are holding onto the flange and the thumb is on the end of the plunger (Figure 1A).</li>
		<li>Press down on plunger in one, continuous motion and inject the full volume of liquid in 6-8 sec.</li>
		<li>Remove needle from vein and stop the bleeding by applying gentle pressure to the tail with a tissue.</li>
		<li>Remove mouse from the restrainer and place mouse into a recovery cage placed on top of a heating pad (bedding should be 37-38 &#176;C). If the injection room is cold, this step is critical for mouse survival. Hold onto the tail of the mouse until bleeding stops completely.</li>
		<li>Observe the mouse for 1 hr following hydrodynamic DNA delivery. An initial period of panting and immobility is normal due to the temporary arrhythmia caused by HGD but make sure that the mouse shows signs of recovery in approximately 5 min. If the breathing of the mouse becomes exceedingly shallow, gently massage the abdomen of the mouse to facilitate breathing. Note that rate of recovery may be slightly influenced by the mouse strain used.</li>
		<li>Return mouse to housing cage and ensure that mouse has an abundant supply of food and water.</li>
	</ol>
	</li>
	<li>Large-volume tail vein injection using an alternative hand position
	<ol>
		<li>Place mouse into the restrainer and prepare for injection by following steps 4.3.1 through 4.3.5, as described previously.</li>
		<li>Rest the non-injecting hand on a flat surface with four fingers bent at a ninety-degree angle. The fingers (all except thumb) should rest on top of each other, creating a platform. Hold the tail of the mouse between the thumb and the pointing finger (Figure 1B).</li>
		<li>Using the other hand, wipe the area to be injected again with an alcohol swab. Allow area to dry.</li>
		<li>Grab syringe with the injecting-hand and hold it by the flange and the end of the plunger, ready for injection.</li>
		<li>Complete the procedure by following this protocol from step 4.3.9 through step 4.3.17, as described previously.</li>
	</ol>
	</li>
</ol>

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J., 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=Haptoglobin-related+protein+is+a+high-affinity+hemoglobin-binding+plasma+protein.">Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein.</a> Blood. 108, 2846-2849 (2006).</li><li>Molina-Portela Mdel, P., Lugli, P., Recio-Pinto, E., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosome+lytic+factor,+a+subclass+of+high-density+lipoprotein,+forms+cation-selective+pores+in+membranes.">Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes.</a> Mol Biochem Parasitol. 144, 218-226 (2005).</li><li>Perez-Morga, D., 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=Apolipoprotein+L-I+promotes+trypanosome+lysis+by+forming+pores+in+lysosomal+membranes.">Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes.</a> Science. 309, 469-472 (2005).</li><li>Molina-Portela, M. P., Samanovic, M., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+of+apolipoprotein+components+within+the+trypanosome+lytic+factor+complex+revealed+in+a+novel+transgenic+mouse+model.">Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model.</a> J Exp Med. 205, 1721-1728 (2008).</li><li>Oli, M. W., Cotlin, L. F., Shiflett, A. M., Hajduk, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+resistance-associated+protein+blocks+lysosomal+targeting+of+trypanosome+lytic+factor+in+Trypanosoma+brucei.">Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei.</a> Eukaryot Cell. 5, 132-139 (2006).</li><li>Thomson, R., Molina-Portela, P., Mott, H., Carrington, M., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery+of+baboon+trypanosome+lytic+factor+eliminates+both+animal+and+human-infective+African+trypanosomes.">Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes.</a> Proc Natl Acad Sci U S A. 106, 19509-19514 (2009).</li><li>Genovese, G., 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=Association+of+trypanolytic+ApoL1+variants+with+kidney+disease+in+African+Americans.">Association of trypanolytic ApoL1 variants with kidney disease in African Americans.</a> Science. 329, 841-845 (2010).</li><li>Tzur, 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=Missense+mutations+in+the+APOL1+gene+are+highly+associated+with+end+stage+kidney+disease+risk+previously+attributed+to+the+MYH9+gene.">Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene.</a> Hum Genet. 128, 345-350 (2010).</li><li>Suda, T., Gao, X., Stolz, D. B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14, 129-137 (2007).</li></ol>

The authors declare that they have no competing financial interests.

When performed correctly, HGD is a remarkably safe and effective means of transgene delivery. The critical steps to successful HGD are: 1) delivering the right amount of DNA in a large volume of saline vehicle 2) into the tail vein of the mouse 3) in less than 8 sec.
Although the process of injection itself undeniably requires some manual dexterity, the success of this six-second procedure often lies in careful preparation of the experiment. The amount of pDNA necessary to achieve maximal gene...

Since its first description by Liu et al. and Zhang et al., hydrodynamic gene delivery (HGD) has become an invaluable tool for studying gene function in rodent model systems1,2. The technique involves the rapid injection (5-7 sec) of a large volume (8-12% body weight) of solution into the tail vein of mice to facilitate the uptake of plasmid DNA by the cells of target organs1,2. These conditions lead to robust gene expression in the liver and less gene expression in the kidney, spleen, lung and heart.
Injection of 10 &#956;g pCMV-LacZ plasmid can transfect as much as 40% of hepatocytes, making HGD the most efficient, non-viral, in vivo gene delivery method to date1. Unlike viral carriers, pDNA is easy to prepare, does not elicit an immune response in the rodent host3 and does not pose a health risk by recombining with endogenous viruses. In addition, since the DNA molecules delivered by HGD do not need packaging, this method is suitable for the delivery of bacterial artificial chromosomes (BAC) as large as 150 kb4. Other types of molecules that have been delivered by a hydrodynamic method include RNA5-10, morpholinos11, proteins12,13 and other small molecules12,14. The advantages and disadvantages of HGD over other delivery methods have been discussed in excellent reviews in the literature15-20 and a number of authors have provided a detailed description of the procedure21-23.
Introducing transgenes into mice by HGD is safe and effective1-3,24 and the method has been used in rats with comparable success25. With certain modifications, proof-of-concept experiments have been carried out in chickens26, rabbits27 and pigs28, although, the in vivo application of this technique in larger animals remains a challenge. When using this method, another common limitation is that many of the available mammalian expression vectors lack the components to achieve a persistent, high level of gene expression. Using a pCMV-Luc plasmid, gene expression in the target organs is evident as early as ten minutes after HGD, however, the initial, high expression level drops sharply in the first week after injection1. Long-term transgene expression is possible depending on the promoter and intron used in plasmid design3,24 however, maintenance of high-level gene expression often requires repeated injections. For this reason, HGD might be less suitable to study chronic diseases that are a result of long-term exposure to damaging proteins or protein products. With these limitations, HGD is an exceptionally powerful tool for studying the potential role of a gene and the effect of it mutants in vivo, as well as the therapeutic effects and regulation of proteins and for establishing animal models of disease (for review, see15). For example, HGD may be used to assign function to domains and amino acids of proteins by individually introducing various gene constructs into mice that have the respective genes knocked out. Furthermore, this technique may be used in any mouse strain.
This protocol describes HGD in mice with a focus on the technical aspects necessary to achieve successful transfection: correct needle insertion into the vein, injection volume and speed of delivery. The application of this method is demonstrated in a mouse model of African trypanosomiasis, a fatal disease of humans and livestock29,30. While several species of trypanosomes cause disease in livestock, most cannot cause disease in humans due to innate immune complexes in blood called trypanosome lytic factors (TLFs)29,31,32. These pore-forming, high-density lipoproteins (HDL) contain two unique, primate-specific proteins: HPR, the ligand, which facilitates the uptake of TLFs into trypanosomes, and APOL-I, the lytic component31,33-38. Trypanosoma brucei rhodesiense is able to infect humans due to expression of a serum resistance-associated protein (SRA) that binds to and neutralizes human APOL-I34,39. Baboon TLF is not neutralized by SRA due to its divergent APOL-I protein40. As reported previously, using a mammalian expression vector (pRG977), transgenic expression of baboon TLF components in mice confers protection against human-infective trypanosomes40. The representative data presented here demonstrate how hydrodynamic gene delivery may be applied to study the therapeutic effects of a protein.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcohol Prep Wipe</td>
			<td style="width:113px;">Webcol</td>
			<td align="right" style="width:119px;">6818</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AST kit, Amplite Colorimetric</td>
			<td style="width:113px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">13801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical Tube, 50 ml</td>
			<td style="width:113px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:113px;">Qiagen</td>
			<td align="right" style="width:119px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:113px;">KIMTECH</td>
			<td align="right" style="width:119px;">34120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse Tail Illuminator</td>
			<td style="width:113px;">Braintree Scientific</td>
			<td style="width:119px;">MTI RST</td>
			<td style="width:167px;">Or equivalent</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 20 Gx 1 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 27 Gx 1/2 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">pRG977 (mammalian expression vector)</td>
			<td style="width:113px;">Regeneron Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Material Transfer Agreement required</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium Chloride, 0.9% INJ USP 10 ml, Hospira&#160;</td>
			<td style="width:113px;">Fisher</td>
			<td style="width:119px;">NC9054335</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Syringe, 3ml, Luer-Lok Tip</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">309657</td>
			<td style="width:167px;">Luer Lock necessary to withstand pressure from HGD</td>
		</tr>
	</tbody>
</table>

transient expression of proteins by hydrodynamic gene delivery in mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

Correct positioning of the needle into the tail vein of the mouse (as illustrated in Figure 1) is a prerequisite for successfully delivering a transgene by hydrodynamics-based transfection. Often the most challenging part of the technique, however, is the retention of the needle within the tail vein without movement so that the entire volume of the injection can be delivered within a 6-8 s period. Minor errors during the injection process may result in vastly reduced transfection efficiency and protein e...

Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

transient-expression-of-proteins-by-hydrodynamic-gene-delivery-in-mice

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28.3K Views.  Hunter College, CUNY. In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Video: Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

Methods Article

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice