S. S. was supported by an American Heart Association postdoctoral fellowship 17POST33670076. K. W. was supported by NIH grants R01 HL138014, R01 HL141256, and R01 HL139819.

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia.
1. Generation and purification of lentivirus particles
NOTE: Lentivirus particles containing the optimized guide RNA can be produced by the detailed protocols provided by Addgene: &#60;https://media.addgene.org/cms/files/Zhang_lab_LentiCRISPR_library_protocol.pdf)&#62;. Optimized methods for high-titer lentivirus preparation and storage are discussed elsewhere17,18. In brief, lentiviruses are produced by co-transfection of a lentivirus vector plasmid, psPAX2, and pMD2.G into HEK 293T cells. Culture supernatant is collected at 48 h post-transfection and concentrated by ultracentrifugation. Lentiviral titer is determined by a commercially available qPCR-based assay. This procedure should be performed in a biosafety class II cabinet.
<ol>
	<li>Prepare a 1:200 solution of collagen (0.0005%) in 1x PBS.</li>
	<li>Coat a 6 well plate with collagen solution and incubate at 37 &#176;C, 5% CO2 for ~30 min.</li>
	<li>Seed 293T cells at a density of 1 x 106 cells per well and incubate at 37 &#176;C, 5% CO2 for ~2 h.</li>
	<li>To prepare the mixture of three transfection plasmids for one well, combine 0.9 &#181;g of lentivirus vector, 0.6 &#181;g of psPAX2, and 0.3&#160;&#181;g of pMD2.G, then achieve a total volume of 10 &#181;L by adding deionized water. Adjust amounts accordingly depending on the number of wells. The amount and ratio of each plasmid may need to be further optimized to suit the researchers needs.</li>
	<li>Carefully add 50 &#181;L of 1x PBS and 5 &#181;L of the diluted PEI MAX (1.0 mg/mL)&#160;to the plasmid mixture and incubate for 15 min at room temperature (RT) (Table 1).</li>
	<li>Add 1 mL of DMEM to the mixture.</li>
	<li>Aspirate media from the 6 well plate, add 1 mL of plasmid mixture, and incubate at 37 &#176;C, 5% CO2 for ~3 h.</li>
	<li>Replace the media with 2 mL of fresh DMEM and incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	<li>Add 1 mL of fresh DMEM and incubate at 37 &#176;C, 5% CO2 for an additional 24 h (total incubation time is 48 h).</li>
	<li>Transfer the culture supernatant to a 50 mL tube and centrifuge at 3,000 x g for 15 min to remove any free-floating cells.</li>
	<li>Filter the supernatant through a 0.45 &#181;m filter.</li>
	<li>Transfer the filtrate to polypropylene centrifuge tubes.</li>
	<li>Ultracentrifuge at 4 &#176;C and 72,100 x g at rmax for 3 h.</li>
	<li>Carefully aspirate the supernatant, leaving behind the white pellet.</li>
	<li>Resuspend the pellet with 100 &#181;L of serum-free hematopoietic cell expansion medium without aeration.</li>
	<li>Keep a 10 &#181;L aliquot to measure the viral titer and store all remaining aliquots at -80 &#176;C until required.</li>
	<li>Titrate the virus with a qPCR-based assay according to the manufacturer&#39;s instructions using the 10 &#181;L viral aliquot.</li>
</ol>
2. Isolation and transduction of lineage-negative cells from mouse bone marrow (Figure 1A)
NOTE: Typically, to isolate enough cells, pairs of tibias, femurs, and humeri are harvested from each mouse. Pelvic and spinal bones may also be harvested as a source of lineage-negative cells.
<ol>
	<li>Isolation of bone marrow cells
	<ol>
		<li>Euthanize 8-10 week old male CRISPR/Cas9 knock-in mice by 5% isoflurane followed by cervical dislocation, then disinfect their skin with 70% ethanol.</li>
		<li>Using dissecting scissors, make a transverse incision in the skin just below the ribcage and peel the skin distally in both directions to expose the legs and arms.</li>
		<li>Carefully separate the lower limbs from the hip bone by dislocating the hip joint. Cut along the femur head to remove the femur completely from the hip. Dislocate the knee and cut at the joint to separate the femur and tibia, while keeping the bone epiphysis intact. Dislocate the ankle joint and peel away the foot and extra muscle.</li>
		<li>Using dissecting scissors, cut over the shoulder to detach the upper limbs. Dislocate the shoulder, then cut at the elbow joint to harvest the humerus bone.</li>
		<li>Use cellulose-fiber wipes to carefully remove muscles from the femurs, tibias, and humeri. Take extra precaution to ensure that the bones do not break during this process.</li>
		<li>Place the isolated bones into a 50 mL conical tube containing RPMI, and place on ice. 
		NOTE: The following steps should be carried out in a biosafety class II cabinet.</li>
		<li>Transfer the bones into a sterile, 100 mm culture dish.</li>
		<li>Grasp the bone with blunt forceps, and using dissecting scissors, carefully cut both epiphyses. 
		NOTE: An insufficient cutting will lead to an incomplete flush of bone marrow, while overly aggressive cutting will result in cell loss.</li>
		<li>Fill a 10 mL syringe with ice-cold RPMI, and using a 22 G needle, flush the bone marrow from the shaft into a new 100 mm culture dish. 
		NOTE: Bones will become white and translucent if the bone shaft has been well-flushed. If not, re-cut the bone ends and flush again.</li>
		<li>After all the bone marrow has been collected, make a single-cell suspension by passing the bone marrow several times through a 10 mL syringe with an 18 G needle. Repeat 10x to ensure a single-cell suspension.</li>
		<li>Filter cell suspension through a 70 &#181;m cell strainer into a 50 mL conical tube.</li>
		<li>Centrifuge at 310 x g for 10 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the cell pellets in an appropriate volume of optimized separation buffer for the following cell separation process.</li>
	</ol>
	</li>
	<li>Isolation and lentivirus transduction of lineage-negative cells 
	&#8203;NOTE: Mouse lineage-negative cells are isolated from the bone marrow of Cas9 transgenic mice3, or other strains of mice, using a lineage depletion kit according to the manufacturer&#39;s instructions. Typically, lineage-negative cells account for 2%-5% of whole bone marrow nucleated cells, and the purity is usually greater than 90% following isolation. The isolated lineage-negative cells are cultured in serum-free hematopoietic cell expansion medium supplemented with 20 ng/mL recombinant murine TPO and 50 ng/mL recombinant murine SCF, then transduced with the lentivirus vector for 16 h at a multiplicity of infection (MOI) = 100.
	<ol>
		<li>To isolate lineage-negative cells, use the lineage cell depletion kit according to the manufacturer&#39;s instructions.</li>
		<li>After isolation resuspend the lineage-negative cells in 1 mL of serum-free hematopoietic cell expansion medium.</li>
		<li>Seed the cells into a 6 well plate at a density of 1.5 x 106 cells/mL (5 x 105 lineage-negative cells/mouse.)</li>
		<li>Add recombinant murine TPO and SCF into wells at final concentrations of 20 ng/mL and 50 ng/mL, respectively.</li>
		<li>Pre-incubate cells at 37 &#176;C in 5% CO2 for ~2 h.</li>
		<li>Add lentivirus at MOI = 100, 4 &#181;g/mL polybrene, and penicillin/streptomycin to the wells and incubate at 37 &#176;C, 5% CO2 for 16-20 h (Figure 1B).</li>
		<li>On the following day, collect the lentivirus transduced cells into a 15 mL conical tube and centrifuge at 300 g for 10 min.</li>
		<li>Carefully aspirate the supernatant and resuspend the pellet in 200 &#181;L of RPMI per mouse. Keep the cells at RT until transplantation into mice (section 3).</li>
	</ol>
	</li>
</ol>
3. Transplantation of transduced cells into lethally irradiated mice
<ol>
	<li>On the day of bone marrow transplantation, place recipient mice into an eight-slice pie cage and expose them to two doses of whole body irradiation (550 Rad/dose, total dose = 1100 Rad), with approximately 4 h between each irradiation session.</li>
	<li>After the second irradiation session, inject transduced lineage-negative cells to each anesthetized recipient mouse via the retro-orbital vein plexus (200 &#181;L in total) using an insulin syringe (Figure 1C).</li>
	<li>After irradiation, mice should be housed in sterilized cages and provided with a soft diet and drinking water supplemented with antibiotics for 14 d.</li>
	<li>At 3-4 weeks after bone marrow transplantation, analyze peripheral blood to check for the engraftment of transduced donor cells (section 4).</li>
</ol>
4. Evaluating the chimerism of peripheral blood
<ol>
	<li>Anesthetize mice with 5% isoflurane and obtain a blood sample from a retro-orbital vein using capillary tubes, and collect it into K2EDTA tubes (the volume in one capillary tube is sufficient for the following assay).</li>
	<li>Transfer 20 &#181;L of blood from the K2EDTA tubes into the 5 mL round bottom polystyrene test tubes, and put on ice.</li>
	<li>Add 1.5 mL of RBC lysis buffer to lyse red blood cells. Incubate for 5 min on ice.</li>
	<li>To neutralize the lysis buffer, wash samples with FACS buffer (1.5 mL/sample).</li>
	<li>Centrifuge at 609 x g at rmax for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Incubate the cells with a cocktail of monoclonal antibodies (diluted in 100 &#181;L FACS buffer/sample) at RT for 20 min in the dark. A complete list of antibodies is provided in the Materials section above.</li>
	<li>Wash the cells once with FACS buffer (2 mL/sample). Centrifuge at 609 x g at rmax (1,800 rpm) for 5 min at 4 &#176;C. Discard the supernatant completely.</li>
	<li>Fix the cells with paraformaldehyde containing fixation buffer (100 &#181;L/tube) for 10 min at 4 &#176;C.</li>
	<li>Wash cells once with FACS buffer (3 mL/sample). Centrifuge at 609 x g at rmax (1,800 rpm) for 5 min at 4 &#176;C. Discard the supernatant completely.</li>
	<li>Suspend the pellet in 400 &#181;L of FACS buffer.</li>
	<li>Keep the samples at 4 &#176;C until analysis by flow cytometry.</li>
</ol>

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The authors have nothing to disclose.

The advantage of this protocol is the creation of animal models harboring specific mutations in hematopoietic cells in a rapid and highly cost-effective manner compared to conventional mouse transgenic approaches. It was found that this methodology enables the generation of mice with hematopoietic cell gene-manipulations within 1 month. There are several critical steps in this protocol that require further consideration.
Screening of gRNA sequence 
...

Many studies in hematology and immunology rely on the availability of genetically modified mice, including conventional and conditional transgenic/knock-out mice that utilize hematopoietic system-specific Cre drivers such as Mx1-Cre, Vav-Cre, and others1,2,3,4,5. These strategies require the establishment of new mouse strains, which can be time-consuming and financially burdening. While revolutionary advances in genome editing technology have enabled the generation of new mouse strains in as few as 3-4 months with the appropriate technical expertise6,7,8,9, much more time is required to amplify the mouse colony before experiments are pursued. In addition, these procedures are costly. For example, Jackson Laboratory lists the current price of knock-out mice generation services at $16,845 per strain (as of December 2018). Thus, methods that are more economical and efficient than conventional murine transgenic approaches are more advantageous.
Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology has led to the development of new tools for rapid and efficient RNA-based, sequence-specific genome editing. Originally discovered as a bacterial adaptive immune mechanism to destroy invading pathogen DNA, the CRISPR/Cas9 system has been used as a tool to increase the effectiveness of genome editing in eukaryotic cells and animal models. A number of approaches have been employed to transmit CRISPR/Cas9 machinery into hematopoietic stem cells (i.e., electroporation, nucleofection, lipofection, viral delivery, and others).
Here, a lentivirus system is employed to transduce cells due to its ability to effectively infect Cas9-expressing murine hematopoietic stem cells and package together the guide RNA expression construct, promoters, regulatory sequences, and genes that encode fluorescent reporter proteins (i.e., GFP, RFP). Using this method, ex vivo gene editing of mouse hematopoietic stem cells has been achieved, followed by successful reconstitution of bone marrow in lethally irradiated mice10. The lentivirus vector employed for this study expresses the Cas9 and GFP reporter genes from the common core EF1a promoter with an internal ribosomal entry site upstream from the reporter gene. The guide RNA sequence is expressed from a separate U6 promoter. This system is then used to create insertion and deletion mutations in the candidate clonal hematopoiesis driver genes Tet2 and Dnmt3a10. However, the transduction efficiency by this method is relatively low (~5%-10%) due to the large size of the vector insert (13 Kbp) that limits transduction efficiency and reduces virus titer during production.
In other studies, it has been shown that larger viral RNA size negatively affects both virus production and transduction efficiency. For example, a 1 kb increase in insert size is reported to decrease virus production by ~50%, and transduction efficiency will decrease to more than 50% in mouse hematopoietic stem cells11. Thus, it is advantageous to reduce the size of the viral insert as much as possible to improve efficiency of the system.
This shortcoming can be overcome by employing Cas9 transgenic mice, in which the Cas9 protein is expressed in either a constitutive or inducible manner12. The constitutive CRISPR/Cas9 knock-in mice expresses Cas9 endonuclease and EGFP from the CAG promoter at the Rosa26 locus in a ubiquitous manner. Thus, a construct with sgRNA under the control of the U6 promoter and RFP reporter gene under the control of the core EF1a promoter can be delivered using the lentivirus vector to achieve genome editing. With this system, the genes of hematopoietic stem cells have been successfully edited, showing a ~90% transduction efficiency. Thus, this protocol provides a rapid and effective method to create mice in which targeted gene mutations are introduced into the hematopoietic system. While our lab is predominantly using this type of technology to study the role of clonal hematopoiesis in cardiovascular disease processes13,14,15, it is also applicable to studies of hematological malignancy16. Furthermore, this protocol can be extended to the analysis of how DNA mutations in HSPC impact other disease or developmental processes in the hematopoietic system.
To establish a robust lentivirus vector system, high titer viral stocks and optimized conditions for the transduction and transplantation of hematopoietic cells are required. In the protocol, instructions are provided on the preparation of a high titer viral stock in section 1, optimizing the culture conditions of murine hematopoietic stem cells in section 2, methods for bone marrow transplantation in section 3, and assessing engraftment in&#160;section 4.

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			<td height="21" style="height:21px;width:515px;">1/2 cc LO-DOSE INSULIN SYRINGE</td>
			<td style="width:159px;">EXELINT</td>
			<td style="width:113px;">26028</td>
			<td style="width:150px;">general supply</td>
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			<td height="21" style="height:21px;">BD Luer-Lok disposable syringes, 10 ml</td>
			<td>BD</td>
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			<td>BD Bioscience</td>
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			<td height="21" style="height:21px;">BD Precisionglide needle, 18 G</td>
			<td>BD</td>
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			<td height="21" style="height:21px;">BD Precisionglide needle, 22 G&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>general supply</td>
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			<td height="21" style="height:21px;width:515px;">BV510-anti-mouse CD8a (Clone 53-6.7)</td>
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			<td>Biolegend</td>
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			<td>CLS3471</td>
			<td>general supply</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:515px;">CRISPR/Cas9 knock-in mice</td>
			<td style="width:159px;">The Jackson Laboratory</td>
			<td style="width:113px;">028555</td>
			<td>mouse</td>
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		<tr height="24">
			<td height="24" style="height:24px;">DietGel 76A</td>
			<td>Clear H2O</td>
			<td>70-01-5022</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) - high glucose</td>
			<td>Sigma Aldrich</td>
			<td>D6429</td>
			<td>Medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;">eBioscience 1X RBC Lysis Buffer</td>
			<td>Thermo fisher Scientific</td>
			<td>00-4333-57</td>
			<td>Solution</td>
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			<td height="21" style="height:21px;">Falcon 100 mm TC-Treated Cell Culture Dish</td>
			<td>Life Sciences</td>
			<td>353003</td>
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			<td height="21" style="height:21px;">Falcon 5 mL round bottom polystyrene test tube</td>
			<td>Life Sciences</td>
			<td>352054</td>
			<td>general supply</td>
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			<td height="21" style="height:21px;">Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td>general supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate&#160;</td>
			<td>Life Science</td>
			<td>353046</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand microhematocrit capillary tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-362566</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand sterile cell strainers, 70 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:515px;">FITC-anti-mouse CD4 (Clone RM4-5)</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:113px;">11-0042-85</td>
			<td>Antibodies</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer</td>
			<td>BD Bioscience</td>
			<td>554655</td>
			<td>Solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Guide-it Compete sgRNA Screening Systems</td>
			<td style="width:159px;">Clontech</td>
			<td style="width:113px;">632636</td>
			<td>Kit</td>
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			<td>Henry Schein</td>
			<td>29404</td>
			<td>Solution</td>
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			<td height="21" style="height:21px;">Lenti-X qRT-PCR Titration Kit&#160;</td>
			<td>Takara</td>
			<td>631235</td>
			<td>Kit</td>
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			<td height="21" style="height:21px;">Lineage Cell Depletion Kit, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>Kit</td>
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			<td>Millipore Sigma</td>
			<td>SLHV004SL</td>
			<td>general supply</td>
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			<td>Gibco</td>
			<td>10010023</td>
			<td>Solution</td>
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			<td height="21" style="height:21px;">PE-Cy7-anti-mouse CD115 (Clone AFS98)</td>
			<td>eBioscience</td>
			<td>25-1152-82</td>
			<td>Antibodies</td>
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			<td height="21" style="height:21px;">PEI MAX</td>
			<td>Polysciences</td>
			<td>24765-1</td>
			<td>Solution</td>
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			<td>Lonza</td>
			<td>17-602F</td>
			<td>Solution</td>
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			<td height="21" style="height:21px;">PerCP-Cy5.5-anti-mouse Ly6G (Clone 1A8)</td>
			<td>BD Biosciences</td>
			<td>560602</td>
			<td>Antibodies</td>
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		<tr height="21">
			<td height="21" style="height:21px;">pLKO5.sgRNA.EFS.tRFP&#160;</td>
			<td>Addgene</td>
			<td>57823</td>
			<td>Plasmid</td>
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		<tr height="21">
			<td height="21" style="height:21px;">pMG2D</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>Plasmid</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Polybrene Infection/Transfection Reagent</td>
			<td>Sigma Aldrich</td>
			<td>TR-1003-G</td>
			<td>Solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene Centrifuge Tubes</td>
			<td>BECKMAN COULTER</td>
			<td>326823</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;">psPAX2&#160;</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Plasmid</td>
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			<td height="21" style="height:21px;">RadDisk &#8211; Rodent Irradiator Disk</td>
			<td>Braintree Scientific</td>
			<td>IRD-P M</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td>Solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine TPO&#160;</td>
			<td>Peprotech&#160;</td>
			<td>315-14</td>
			<td>Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemSpan SFEM</td>
			<td>STEMCELL Technologies</td>
			<td>09600</td>
			<td>Solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TOPO TA cloning kit for sequencing with One Shot TOP10 Chemically Competent E.coli</td>
			<td style="width:159px;">Thermo fisher Scientific</td>
			<td>K457501</td>
			<td>Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zombie Aqua Fixable Viability Kit</td>
			<td>BioLegend</td>
			<td>423102</td>
			<td>Solution&#160;</td>
		</tr>
	</tbody>
</table>

lentiviral crispr/cas9-mediated genome editing for the study of hematopoietic cells in disease models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Using the above described protocol, approximately 0.8-1.0 x 108 bone marrow cells per mouse have been obtained. The number of lineage-negative cells we obtain is approximately 3 x 106 cells per mouse. Typically, the yield of bone marrow lineage-negative cells is 4%-5% of that of total bone marrow nuclear cells.
Chimerism of transduced cells (RFP-positive) is evaluated by flow cytometry of the peripheral blo...

Watch this Scientific Journal Video about Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models at JoVE.com

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

lentiviral-crisprcas9-mediated-genome-editing-for-study-hematopoietic

Research

JoVE Journal

Immunology and Infection

12.0K Views.  University of Virginia School of Medicine. Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications. 

Video: Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.