These studies were partially funded by a grant from the George F. Haddix Fund of Creighton University (KMD). Figure 1 is generated in biorender.com, and Figure 3 is generated in benchling.com.

The details of the species, reagents, and equipment used in this study are listed in the Table of Materials. The sequence of the 17,004 base pair pGRUB construct is provided in Supplementary File 1.
1. Culturing trophozoites
<ol>
	<li>Thaw frozen N. gruberi trophozoites at 37 &#176;C for 3 min.</li>
	<li>Inoculate trophozoites into T25 flasks in modified peptone/yeast extract/nucleic acid/folic acid/hemin with 10% fetal calf serum (PYNFH) media plus 2% buffer (disodium phosphate, potassium dihydrogen phosphate).</li>
	<li>Culture the trophozoites at 25&#160;&#176;C until ~80% confluent, where they exhibit an amoeboid morphology (Figure 2A). 
	NOTE: When initially reviving a culture of trophozoites from their frozen state, it can take up to 5-7 days for a T25 to become confluent. Decant media every 3-4 days and replace with fresh media.</li>
	<li>Place the confluent flasks on ice for 5-10 min to release the adhered trophozoites from the plastic. Gently swirl the flask to dislodge any remaining adherent cells. The trophozoites now have a &#39;rounded up&#39; morphology (Figure 2B).</li>
	<li>Split the cultures 1:2 to 1:4 into new tissue culture flasks.</li>
	<li>Remove the PYNFH and replace it with sterile encystment media (120 mM sodium chloride, 0.03 mM magnesium chloride, 1 mM disodium phosphate, 1 mM potassium dihydrogen phosphate, 0.03 mM calcium chloride, 0.02 mM iron chloride, pH 6.8)22 to encyst the N. gruberi. 
	NOTE: The majority of trophozoites become encysted by 72 h (Figure 2C). Excystment should be completed within 72 h.</li>
	<li>Remove cysts from flasks, centrifuge at 600 x g for 10 min at 20&#160;&#176;C, and resuspend cysts in PYNFH media with 10% fetal calf serum to excyst the cells. Incubate at 25&#160;&#176;C for 24 h until trophozoites begin to emerge.</li>
</ol>
2. Transfecting trophozoites
<ol>
	<li>Plate the trophozoites into a 6-well flat-bottomed tissue culture plate (1 mL of 1 x 106 trophozoites/mL) and culture at 25&#160;&#176;C.</li>
	<li>Transfect the cells when they reach 70%-80% confluent (~10.4 x 104 cells/cm2 in 6-well clusters). Warm the transfection reagent to room temperature before use.</li>
	<li>Prepare the plasmid-transfection reagent mixture as follows: 75 ng of plasmid DNA and 0.225 &#181;L of the transfection reagent in 200 &#181;L of PYNFH media without FBS. Prepare sufficient plasmid-transfection reagent mixture to perform transfection in duplicate. 
	NOTE: A bacterial plasmid containing a full-length clone of N. gruberi CERE (pGRUB) was used in this study. Empty plasmid (pGEM) was used as a negative control (Figure 3).</li>
	<li>Incubate the plasmid-transfection reagent mixture at room temperature for 10 min.</li>
	<li>Remove ~800 &#181;L of medium from the trophozoite monolayers just prior to transfection.</li>
	<li>Put the plasmid-transfection reagent mixture on the trophozoites.</li>
	<li>Gently swirl the plate every 15 min to prevent the media from aggregating at the edges of the plate.</li>
	<li>Wash the monolayers once with 2 mL of growth medium after 1 h incubation at 25&#160;&#176;C.</li>
	<li>Treat the monolayers with 10 U DNase in 1 mL of 10x DNase buffer for 15 min at 37&#160;&#176;C to remove residual plasmid DNA, wash once with growth medium, add 2 mL of fresh media, and place at 25&#176;C. Incubate the plate for 24-36 h.</li>
	<li>Place the plate on ice for 10 min to release cells.</li>
	<li>Split one well (1:2) into two&#160;new wells.</li>
	<li>Collect trophozoites from the remaining wells for experimental purposes. 
	NOTE: All cultures are performed in duplicate. For each condition, 1 well is used for passage, and 1 is used for CERE isolation.</li>
</ol>
3. Isolating CERE from Naegleria
<ol>
	<li>Calculate the number of trophozoites in each well used for CERE isolation by trypan blue exclusion staining and a hemocytometer23.</li>
	<li>Place the trophozoites in a 2.0 mL tube.</li>
	<li>Wash trophozoites once in 100 mM sodium chloride (pH 7.5) via centrifugation (600 x g, 10 min, 4 &#176;C). 
	NOTE: Sodium chloride prevents the trophozoites from adhering to the sides of the tube.</li>
	<li>Remove the supernatant and resuspend cell pellets in 100 &#181;L of 100 mM EDTA (pH 7.5).</li>
	<li>Place the tube in a heat block for 15 min at 98&#160;&#176;C to lyse the cells and inactivate the enzymes in the trophozoites. 
	NOTE: This step inactivates DNases, which are abundant in Naegleria trophozoites. DNases may interfere with the ability to detect native CERE and the molecular clone in step 4. DNA yield is decreased when over ~3 x 106 cells are used in step 3.4.</li>
	<li>Centrifuge the tubes at top speed (16,000 x g) at 4&#160;&#176;C for 10 min to pellet the debris.</li>
	<li>Transfer the supernatants to a new tube.</li>
	<li>Ethanol precipitate the supernatants with 0.5 volumes of 7.5 M ammonium acetate (pH 7.5), 3 volumes of absolute ethanol, and 1 &#181;L of 10 mg/mL glycogen as a carrier.</li>
	<li>Invert the tube several times and place the tube at -80&#160;&#176;C for at least 1&#160;h or overnight.</li>
	<li>Warm the samples to room temperature.</li>
	<li>Centrifuge the tubes at room temperature for 10 min at 16,000 x g.</li>
	<li>Remove the supernatant and wash the pellet with 70% ethanol by centrifugation for 5 min at 16,000 x g (at room temperature).</li>
	<li>Remove ethanol and air-dry the pellet for 5 min.</li>
	<li>Resuspend the dried pellet in sterile deionized (DI) water. Normalize volume to 10,000 trophozoites per &#181;L.</li>
	<li>Store at -80&#160;&#176;C until use in PCR.</li>
</ol>
4. PCR detection of transformed trophozoites
<ol>
	<li>Use 20,000 cell equivalents of DNA, 600 nM primers (Table 1), and Taq polymerase and perform the PCR24. 
	NOTE: Primers that distinguish between native CERE and the cloned CERE are listed in Table 1, as are the PCR conditions. Figure 3 shows the location of the primers on the constructs. PCR will require optimization depending on the primers used as well as the specific PCR mix utilized.</li>
	<li>Microwave 0.8% agarose in 1x Tris base, acetic acid, and EDTA (TAE) buffer until the agarose is dissolved.</li>
	<li>Cool the agarose to ~50&#160;&#176;C at room temperature.</li>
	<li>Pour the solution into a gel casting tray containing a comb to form wells.</li>
	<li>Let the gel sit at room temperature until solidified.</li>
	<li>Place the gel in the electrophoresis apparatus.</li>
	<li>Decant 1x TAE into the electrophoresis chamber until the gel is covered.</li>
	<li>Remove the comb.</li>
	<li>Add 6x loading dye (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol) to samples and load the gel. Use a DNA ladder to determine the size of the PCR product.</li>
	<li>Attach the electrode, turn on the power supply, and run the gel at 80 V for ~1.5-2 h.</li>
	<li>Turn off the power and remove the gel.</li>
	<li>Stain the gel with DNA dye (e.g., 10 &#181;L of 10 mg/mL ethidium bromide) in 100 mL of DI water for 10 min.</li>
	<li>Destain the gel for 20 min in DI water.</li>
	<li>Visualize gel on an ultraviolet (UV) light system and document the results.</li>
</ol>

Using a CERE-Based Vector for Transfection of Naegleria gruberi Trophozoites

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No financial conflicts of interest were declared.

The protocol outlined herein is very straightforward, although every construct will likely require some degree of optimization, particularly of the DNA-transfection reagent ratio, depending on the nature of the construct and the species of Naegleria used. We have only tested one commercially available transfection reagent using this protocol, but it is likely that several others may be effective. Given that a full-length clone of the CERE is used, containing a functional origin of replication and thus replicatin...

The Naegleria genus contains over 45 species, although it is unlikely that all members of the species have been identified1. Naegleria can exist in different forms: as trophozoites (amoebae), as flagellates, or, when resources are severely limited, as cysts1,2,3,4. The Naegleria genus is recognized for its one particularly dangerous species, Naegleria fowleri, known as &#39;the brain-eating amoeba&#39; (reviewed in1,2,3,4,5,6,7), which is the cause of the almost universally fatal primary amoebic meningoencephalitis.
Naegleria encode their rDNA on the CERE located in the nucleolus. Complete sequencing of three Naegleria species&#39; genomes confirms the absence of rDNA in the nuclear genome8,9,10,11. Based on the limited full-length CERE sequences, the CERE ranges from approximately 10 kbp to 18 kbp in length. CERE of each species of Naegleria carry a single rDNA cistron (containing the 5.8, 18, and 28S ribosomal DNA) on each of approximately 4,000 CERE per cell12,13,14. Other than rDNA, each CERE has a large non-rDNA sequence (NRS). CERE sizes vary between species; the differences are mainly due to the varying length of the NRS, as the rDNA sequences are highly conserved across the genus1,15,16,17,18,19,20. The mapping of a single origin of DNA replication within the N. gruberi CERE NRS21 provides strong support for the hypothesis that Naegleria CERE replicates independently of the nuclear genome.
N. gruberi is a non-pathogenic amoeba often used to study Naegleria biology. We developed a methodology for transforming N. gruberi trophozoites with a CERE clone from the same species to test the hypothesis that Naegleria amoebae tolerate and independently replicate CERE within the trophozoites. This was accomplished by transforming N. gruberi with a full-length clone of N. gruberi CERE into trophozoites and following the fates of the donor CERE clone by polymerase chain reaction (PCR). A general overview of the protocol is outlined in Figure 1. The data presented herein demonstrate that the donor clone can be detected through at least seven passages in tissue culture, as well as be maintained through encystment and excystment. These studies form the basis for a means to dissect how CERE replicates, as well as explore its use as a vector to transfect Naegleria.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Bio Rad</td>
			<td>161-3102</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma Aldrich</td>
			<td>A-7330</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C-4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Crushed ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Flasks, T-75</td>
			<td>Thermo Scientific</td>
			<td>130190</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Plate, 6-well</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma Aldrich</td>
			<td>D-4527</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M</td>
			<td>Affymetrix</td>
			<td>15694</td>
			<td></td>
		</tr>
		<tr>
			<td>Electropheresis Gel Apparatus</td>
			<td>Amersham Biosciences</td>
			<td>80-6052-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes, 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes, 2 mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma Aldrich</td>
			<td>E-8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic Acid</td>
			<td>Sigma Aldrich</td>
			<td>F7876-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb Plus Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM1331</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific</td>
			<td>UN2789</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Green PCR Master Mix</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Block</td>
			<td>Thermo Scientific</td>
			<td>88871001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma Aldrich</td>
			<td>51280</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron Chloride</td>
			<td>Sigma Aldrich</td>
			<td>372870-256</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligase</td>
			<td>NEB</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Magneisum Chloride</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Thermo Scientific</td>
			<td>MySpin 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>TMS</td>
			<td></td>
		</tr>
		<tr>
			<td>N. gruberi</td>
			<td>ATCC</td>
			<td>30224</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic Acid</td>
			<td>Chem Impex Int&#8217;l</td>
			<td>#01625</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Gibco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM</td>
			<td>Promega</td>
			<td>P2251</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate</td>
			<td>Sigma Aldrich</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac HC Electropharesis Power Supply Unit</td>
			<td>Bio Rad</td>
			<td>1645052</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MCB Reagents</td>
			<td>SX0420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate, dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S2554</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop Centrifuge</td>
			<td>eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris, base</td>
			<td>Sigma Aldrich</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaFect Reagent</td>
			<td>Promega</td>
			<td>E4981</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Scale</td>
			<td>Denver Instruments</td>
			<td>APX-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Gibco</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfection of a molecular clone of naegleria gruberi rdna into n. gruberi trophozoites

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

PCR of trophozoites that have been transfected with pGRUB demonstrates that the transfected CERE is detected through at least seven passages of the trophozoites, as well as encystment and excystment (Figure 4). The primers used in the PCR anneal to both the pGEM vector and the CERE sequence, thereby ensuring that the PCR does not detect native CERE. PCR following transfection of pGEM into trophozoites indicated that pGEM was negative (Figure 4), indicating that ...

Author Spotlight: Understanding the Propagation of Closed Circular Extrachromosomal rDNA Containing Element (CERE) in Naegleria gruberi

This protocol describes a methodology to transfect Naegleria gruberi trophozoites with a construct that is maintained throughout passaging trophozoites in vitro, as well as through encystment and excystment.

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While ...

transfection-molecular-clone-naegleria-gruberi-rdna-into-n-gruberi

Research

JoVE Journal

Biology

471 Views.  Creighton University School of Medicine. This protocol describes a methodology to transfect Naegleria gruberi trophozoites with a construct that is maintained throughout passaging trophozoites in vitro, as well as through encystment and excystment.

Video: Author Spotlight: Understanding the Propagation of Closed Circular Extrachromosomal rDNA Containing Element (CERE) in Naegleria gruberi

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

Protein Transfection of Mouse Lung

Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes1. In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice2,3 or viral or non-viral vectors that elevate protein levels via increased gene expression4. Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model5. While conditional transgenics avert problems associated with chronic gene expression6, the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement7. As with transgenics, the use of viral and non-viral vectors is expensive8 and can provoke dose-dependent inflammatory responses that confound results9 and hinder expression10. Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector11,12. Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung13.
Using &beta;-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 &mu;l of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice14. The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity15. Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27&plusmn;4 control vs. 31&plusmn;5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.

Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

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.