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Method Article

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

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DOI:

10.3791/51481

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May 5th, 2014

 , 
1Department of Biological Sciences, Hunter College, CUNY

In This Article

Summary

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.

Abstract

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).

Introduction

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 μg 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.

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Protocol

All experiments described here were approved by the Institutional Animal Care and Use Committee of Hunter College, City University of New York.

1. Preparation of endotoxin-free plasmid DNA

  1. Pick a single colony of bacteria containing the gene of interest in a mammalian expression vector from a freshly streaked selective plate.
  2. Follow the recommendations found in the handbook of a commercially available endotoxin-free plasmid purification kit for growing and harvesting bacteria.
  3. Purify the endotoxin-free plasmid DNA from bacterial cells by following the protocol of a commercially available endotoxin-free plasmid purification kit.
  4. 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.
  5. 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.
    1. Clean the lower and upper optical surfaces of the micro spectrophotometer sample retention system as follows. Pipette 1 μl 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.
    2. Open the software of the micro spectrophotometer and select the nucleic acids module.
    3. Place 1 μl clean deionized water on the lower optical system, lower the lever arm and select “initialize” from the program software. Once initialization is complete, clean both optical surfaces with a tissue..
    4. Perform blank measurement by loading 1 μl of endotoxin-free TE buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA) and selecting “blank” from the program software. Once blank measurement is done, clean both optical surfaces with a tissue.
    5. Perform sample measurement by loading 1 μl of endotoxin-free DNA sample and selecting “measure” 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.

2. Weighing of mice

  1. Mark mice in a manner appropriate for the strain. Note: marking of the tail is not recommended for this procedure.
    1. Mark mouse species that have white fur (e.g. Swiss Webster) on their backs with a black marker. Reapply marks over time as necessary.
    2. Mark mouse species that have black fur (e.g. C57BL/6) by ear punching several days in advance.
  2. 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.

3. Preparation of syringes

  1. Preparation of the injection mix
    1. Calculate the amount of DNA needed by assuming 5 - 50 μg endotoxin-free plasmid DNA for the injection of each mouse.
      1. Determine the optimal amount of plasmid DNA experimentally.
      2. 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 μg plasmid DNA per mouse. To determine the amount of DNA needed in this case, calculate with 5 mice per group: 5 x 50 μg = 250 μg DNA needed for each group.
    2. Determine the amount of saline (0.9% sodium chloride) needed by assuming 10% of body weight for the injection of each mouse.
      1. According to the above example, inject each 25 g mouse with 50 μg of plasmid DNA in 2.5 ml saline (2.5 g liquid).
      2. 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.
    3. Use preservative- and endotoxin-free, sterile saline that has been approved for human use, in 10 ml, multiple use vials.
    4. 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.
    5. 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 μg of control and 250 μg of experimental plasmid DNA into separate conical tubes.
    6. 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).
    7. Allow all solutions to reach room temperature prior to injection.
  2. Preparation of syringes
    1. 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.
    2. 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.
    3. 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.

4. Hydrodynamic DNA delivery

  1. 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 °C or if using anesthesia, place the mice on a heating pad set at 37 °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.
  2. Warm the mice to dilate blood vessels.
    1. 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 °C. The temperature should be low enough to keep the mice on the heat pad for an extended period.
    2. 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.
    3. 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.
  3. Large-volume tail vein injection
    1. Immobilize a dorsal access restrainer on a flat surface by laboratory tape.
    2. 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.
    3. 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.
    4. 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.
    5. Wipe the tail of the mouse thoroughly with an alcohol swab.
    6. 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)
    7. Using the other hand, wipe the area to be injected again with an alcohol swab. Allow area to dry.
    8. 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.
    9. 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.
    10. 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.
    11. 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).
    12. 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).
    13. Press down on plunger in one, continuous motion and inject the full volume of liquid in 6-8 sec.
    14. Remove needle from vein and stop the bleeding by applying gentle pressure to the tail with a tissue.
    15. Remove mouse from the restrainer and place mouse into a recovery cage placed on top of a heating pad (bedding should be 37-38 °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.
    16. 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.
    17. Return mouse to housing cage and ensure that mouse has an abundant supply of food and water.
  4. Large-volume tail vein injection using an alternative hand position
    1. Place mouse into the restrainer and prepare for injection by following steps 4.3.1 through 4.3.5, as described previously.
    2. 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).
    3. Using the other hand, wipe the area to be injected again with an alcohol swab. Allow area to dry.
    4. Grab syringe with the injecting-hand and hold it by the flange and the end of the plunger, ready for injection.
    5. Complete the procedure by following this protocol from step 4.3.9 through step 4.3.17, as described previously.

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Results

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...

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Discussion

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...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Alcohol Prep WipeWebcol6818
AST kit, Amplite ColorimetricAAT Bioquest13801
Conical Tube, 50 mlBD Falcon352070
EndoFree Plasmid Maxi KitQiagen12362
KimWipesKIMTECH34120
Mouse Tail IlluminatorBraintree ScientificMTI RSTOr equivalent
Needle, 20 Gx 1 (0.9mmx25mm)BD305175
Needle, 27 Gx 1/2 (0.9mmx25mm)BD305109
pRG977 (mammalian expression vector)Regeneron PharmaceuticalsMaterial Transfer Agreement required
Sodium Chloride, 0.9% INJ USP 10 ml, Hospira FisherNC9054335
Syringe, 3ml, Luer-Lok TipBD309657Luer Lock necessary to withstand pressure from HGD

References

  1. Liu, F., Song, Y., Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258-1266 (1999).
  2. Zhang, G., Budker, V., Wolff, J. A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther. 10, 1735-1737 (1999).
  3. Miao, C. H., Thompson, A. R., Loeb, K., Ye, X. Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther. 3, 947-957 (2001).
  4. Magin-Lachmann, C., et al. In vitro and in vivo delivery of intact BAC DNA -- comparison of different methods. J Gene Med. 6, 195-209 (2004).
  5. Chang, J., Sigal, L. J., Lerro, A., Taylor, J. Replication of the human hepatitis delta virus genome Is initiated in mouse hepatocytes following intravenous injection of naked DNA or RNA sequences. J Virol. 75, 3469-3473 (2001).
  6. Giladi, H., et al. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther. 8, 769-776 (2003).
  7. Kobayashi, N., et al. Vector-based in vivo RNA interference: dose- and time-dependent suppression of transgene expression. J Pharmacol Exp Ther. 308, 688-693 (2004).
  8. Layzer, J. M., et al. In vivo activity of nuclease-resistant siRNAs. Rna. 10, 766-771 (2004).
  9. McCaffrey, A. P., et al. RNA interference in adult mice. Nature. 418, 38-39 (2002).
  10. McCaffrey, A. P., et al. Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice. Mol Ther. 5, 676-684 (2002).
  11. McCaffrey, A. P., Meuse, L., Karimi, M., Contag, C. H., Kay, M. A. A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology. 38, 503-508 (2003).
  12. Zhang, G., et al. Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther. 11, 675-682 (2004).
  13. Kobayashi, N., Kuramoto, T., Yamaoka, K., Hashida, M., Takakura, Y. Hepatic uptake and gene expression mechanisms following intravenous administration of plasmid DNA by conventional and hydrodynamics-based procedures. J Pharmacol Exp Ther. 297, 853-860 (2001).
  14. Kobayashi, N., Nishikawa, M., Hirata, K., Takakura, Y. Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery. J Gene Med. 6, 584-592 (2004).
  15. Bonamassa, B., Hai, L., Liu, D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 28, 694-701 (2011).
  16. Al-Dosari, M. S., Gao, X. Nonviral gene delivery: principle, limitations, and recent progress. Aaps J. 11, 671-681 (2009).
  17. Gao, X., Kim, K. S., Liu, D. Nonviral gene delivery: what we know and what is next. Aaps J. 9, (2007).
  18. Herweijer, H., Wolff, J. A. Gene therapy progress and prospects: hydrodynamic gene delivery. Gene Ther. 14, 99-107 (2007).
  19. Kobayashi, N., Nishikawa, M., Takakura, Y. The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels. Adv Drug Deliv Rev. 57, 713-731 (2005).
  20. Al-Dosari, M. S., Knapp, J. E., Liu, D. Hydrodynamic delivery. Adv Genet. 54, 65-82 (2005).
  21. Bell, J. B., et al. Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection. Nat Protoc. 2, 3153-3165 (2007).
  22. Osorio, F. G., de la Rosa, J., Freije, J. M. Luminescence-based in vivo monitoring of NF-kappaB activity through a gene delivery approach. Cell Commun Signal. 11, (2013).
  23. Knapp, J. E., Liu, D. Hydrodynamic delivery of DNA. Methods Mol Biol. 245, 245-250 (2004).
  24. Yang, J., Chen, S., Huang, L., Michalopoulos, G. K., Liu, Y. Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth. Hepatology. 33, 848-859 (2001).
  25. Maruyama, H., et al. High-level expression of naked DNA delivered to rat liver via tail vein injection. J Gene Med. 4, 333-341 (2002).
  26. Hen, G., et al. Expression of foreign genes in chicks by hydrodynamics-based naked plasmid transfer in vivo. Domestic animal endocrinology. 30, 135-143 (2006).
  27. Eastman, S. J., et al. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA. Hum Gene Ther. 13, 2065-2077 (2002).
  28. Yoshino, H., Hashizume, K., Kobayashi, E. Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume. Gene Ther. 13, 1696-1702 (2006).
  29. Vanden Bossche, P. Some general aspects of the distribution and epidemiology of bovine trypanosomosis in southern Africa. Int J Parasitol. 31, 592-598 (2001).
  30. Fevre, E. M., Picozzi, K., Jannin, J., Welburn, S. C., Maudlin, I. Human African trypanosomiasis: Epidemiology and control. Adv Parasitol. 61, 167-221 (2006).
  31. Lugli, E. B., Pouliot, M., Portela Mdel, P., Loomis, M. R., Raper, J. Characterization of primate trypanosome lytic factors. Mol Biochem Parasitol. 138, 9-20 (2004).
  32. Pays, E., et al. The trypanolytic factor of human serum. Nat Rev Microbiol. 4, 477-486 (2006).
  33. Raper, J., Fung, R., Ghiso, J., Nussenzweig, V., Tomlinson, S. Characterization of a novel trypanosome lytic factor from human serum. Infect Immun. 67, 1910-1916 (1999).
  34. Vanhamme, L., et al. Apolipoprotein L-I is the trypanosome lytic factor of human serum. Nature. 422, 83-87 (2003).
  35. Nielsen, M. J., et al. Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein. Blood. 108, 2846-2849 (2006).
  36. Molina-Portela Mdel, P., Lugli, P., Recio-Pinto, E., Raper, J. Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes. Mol Biochem Parasitol. 144, 218-226 (2005).
  37. Perez-Morga, D., et al. Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes. Science. 309, 469-472 (2005).
  38. Molina-Portela, M. P., Samanovic, M., Raper, J. Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model. J Exp Med. 205, 1721-1728 (2008).
  39. Oli, M. W., Cotlin, L. F., Shiflett, A. M., Hajduk, S. L. Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei. Eukaryot Cell. 5, 132-139 (2006).
  40. Thomson, R., Molina-Portela, P., Mott, H., Carrington, M., Raper, J. Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes. Proc Natl Acad Sci U S A. 106, 19509-19514 (2009).
  41. Genovese, G., et al. Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science. 329, 841-845 (2010).
  42. Tzur, S., et al. Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene. Hum Genet. 128, 345-350 (2010).
  43. Suda, T., Gao, X., Stolz, D. B., Liu, D. Structural impact of hydrodynamic injection on mouse liver. Gene Ther. 14, 129-137 (2007).

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Transient Protein ExpressionNaked Plasmid DNATail Vein InjectionWestern Blot AnalysisMouse Blood SamplingPlasmid DNA PreparationSyringe Loading TechniqueNeedle Position MaintenanceAPOL-I HPR Detection
Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
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