Name: lentiviral crispr/cas9-mediated genome editing for the study of hematopoietic cells in disease models
Uploaded: 2019-10-03T13:45:00
Description: Watch this Scientific Journal Video about Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models at JoVE.com

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 prepara...

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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>
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			<td height="21" style="height:21px;">APC-anti-mouse Ly6C (Clone AL-21)</td>
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			<td height="21" style="height:21px;">BD Microtainer blood collection tubes, K2EDTA added</td>
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			<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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			<td height="21" style="height:21px;">eBioscience 1X RBC Lysis Buffer</td>
			<td>Thermo fisher Scientific</td>
			<td>00-4333-57</td>
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			<td height="21" style="height:21px;">Falcon 100 mm TC-Treated Cell Culture Dish</td>
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			<td height="21" style="height:21px;">Falcon 50 mL Conical Centrifuge Tubes</td>
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			<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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			<td height="21" style="height:21px;">Fisherbrand sterile cell strainers, 70 &#956;m</td>
			<td>Fisher Scientific</td>
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			<td height="21" style="height:21px;width:515px;">FITC-anti-mouse CD4 (Clone RM4-5)</td>
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			<td style="width:113px;">11-0042-85</td>
			<td>Antibodies</td>
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			<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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		<tr height="21">
			<td height="21" style="height:21px;">Isothesia (Isoflurane) solution</td>
			<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 height="21" style="height:21px;">Millex-HV Syringe Filter Unit, 0.45 mm</td>
			<td>Millipore Sigma</td>
			<td>SLHV004SL</td>
			<td>general supply</td>
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		<tr height="21">
			<td height="21" style="height:21px;">PBS pH7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Solution</td>
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		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;">PEI MAX</td>
			<td>Polysciences</td>
			<td>24765-1</td>
			<td>Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin Mixture</td>
			<td>Lonza</td>
			<td>17-602F</td>
			<td>Solution</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMG2D</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>Plasmid</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene Centrifuge Tubes</td>
			<td>BECKMAN COULTER</td>
			<td>326823</td>
			<td>general supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX2&#160;</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Plasmid</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td>Solution</td>
		</tr>
		<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 ...

We've developed an efficient and economical method to establish mice with gene manipulation specifically in hematopoietic stem cells, Using genome editing technology and lentivirus-mediated transgene delivery systems. The advantage of this protocol is that we can create animal models harboring specific mutations in hematopoietic cells in a rapid and cost-effective manner compared to conventional mouse transgenic approaches. Demonstrating the procedure will be Ying Wang, a researcher from my laboratory.To generate lentivirus particles, start by coding a six well plate with collagen solution and incubating it at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. Then, seed 293T cells onto the plate and incubate it for another two hours. Meanwhile, prepare the mixture of three transfection plasmids by combining 0.9 micrograms of lentivirus vector, 0.6 micrograms of psPAX2 and 0.3 micrograms of pMD2.G and deionized to 10 microliters per well. Then, carefully add 50 microliters of 1X PBS and five microliters of diluted PEI MAX to the plasmid mixture, and incubate the mixture at room temperature for 15 minutes. After the incubation, add one milliliter of DMEM.Aspirate the media from the six well plate and add one milliliter of plasmid mixture to each well. Incubate the plate for another three hours. Replace the media with fresh DMEM and incubate for 24 hours.Add one milliliter of fresh DMEM and incubate an additional 24 hours. After total 48 hour incubation, transfer the supernatant to a 50 milliliter tube and centrifuge it at 3000 times G for 15 minutes to remove any free-floating cells. Filter the supernatant through a 0.45 micrometer filter and ultra-centrifuge it for three hours.Carefully aspirate the supernatant, leaving behind the white pellet. Re-suspend the pellet with 100 microliters of serum-free hematopoietic cell expansion medium without aeration. Keep a 10 microliter aliquot to measure the viral titer and store the rest of the suspension at minus 80 Celsius until ready to use.After isolating the bones, place them into a 50 milliliter conical tube with RPMI and place the tube on ice. In a bio-safety cabinet, transfer the bones into a sterile 100 millimeter culture dish. Then grasp a bone with blunt forceps, and use dissection scissors to carefully cut both epiphyses.Fill a 10 milliliter syringe with ice cold RPMI and use a 22 gauge needle to flush the bone marrow from the shaft into a new 100 millimeter culture dish. After all the bone marrow has been collected, make a single cell suspension by passing the bone marrow through a 10 milliliter syringe with an 18 gauge needle. Filter the cell suspension through a 70 micrometer cell strainer into a 50 milliliter conical tube.Then centrifuge the tube according to the manuscript directions and aspirate the supernatant. Re-suspend the cell pellets in an appropriate volume of optimized separation buffer. To isolate lineage negative cells, use a lineage cell depletion kit according to the manufacturer's instructions.Re-suspend the isolated lineage negative cells in serum-free hematopoietic cell expansion medium. Pre-incubate the cells at 37 degrees celsius in 5%carbon dioxide for two hours. Then add lentivirus according to the manuscript directions and leave the cells in the incubator for another 16 to 20 hours.The next day, collect the lentivirus-transduced cells in a 15 milliliter conical tube and centrifuge them at 300 times G for 10 minutes. According to NHA guidelines, it is important to handle lentivirus vectors at Reece Group level two. Precautions must be taken, based on facility regulations.Carefully aspirate the supernatant and re-suspend the pellet in 200 microliters of RPMI per mouse. Keep the cells at room temperature until transplantation into mice. After the second irradiation session, retro-orbitally inject transduced delineage negative cells into each anesthetized mouse using an insulin syringe.Three to four weeks after bone marrow transplantation, obtain blood samples from the retro-orbital vein using capillary tubes. Transfer 20 microliters of blood into round-bottom polystyrene test tubes and place on ice. After RBC lysis, incubate the cells with a cocktail of monoclonal antibodies in the dark for 20 minutes at room temperature.After incubation, wash, fix and centrifuge the cells and re-suspend the cell pellet in 400 microliters of FACS buffer. Keep the samples at four degrees Celsius until analysis by flow cytometry. Success of lineage negative cell transduction can be evaluated with flow cytometric detection of RFP.It has been demonstrated that after seven days of in vitro culture, on average, 75.7%of cells are transduced. Flow cytometry has also been used to analyze mouse peripheral blood after reconstitution with transduced and non-transduced hematopoietic stem cells. An average of 94.8%of neutrophils, 93.5%of monocytes, and 82.7%of B cells express RFP.Genomic DNA from the RFP-positive blood cells has been PCR amplified and sub-cloned for sequence analysis. Various mutations are detected and 69%of the clones show out of frame or premature stop mutations. To succeed with this procedure, preparation of high antivirus stock and optimized conditions for transduction and transplantation of hematopoietic cells are required.We have applied this technique to study the law of in cardio-vascular disease. But it is also useful to study hematological malignancy. This method enables us to study the functions of new types of genes in hematopoietic system in high efficient and high manner.We gather this over the availability of transgenic mice.

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

Research

JoVE Journal

Immunology and Infection

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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.

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 ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...