The authors want to acknowledge the staff of the flow cytometry facility and animal facility of CSCR. A. C. is funded by an ICMR-SRF fellowship, K. V. K. is funded by a DST-INSPIRE fellowship, and P. B. is funded by a CSIR-JRF fellowship. This work was funded by the Department of Biotechnology, Government of India (grant no. BT/PR26901/MED/31/377/2017 and BT/PR31616/MED/31/408/2019)

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivector-mediated+clonal+tracking+reveals+intrinsic+heterogeneity+in+the+human+hematopoietic+stem+cell+compartment+and+culture-induced+stem+cell+impairment.">Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment.</a> Blood. 103 (2), 545-552 (2004).</li><li>Piras, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+escape+innate+sensing+but+trigger+p53+in+human+hematopoietic+stem+and+progenitor+cells.">Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells.</a> EMBO Molecular Medicine. 9 (9), 1198-1211 (2017).</li><li>Christopher, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+expansion+of+human+CD34+CD133+CD90++hematopoietic+stem+cells+enhances+gene-modified+cell+frequency+for+gene+therapy.">Preferential expansion of human CD34+CD133+CD90+ hematopoietic stem cells enhances gene-modified cell frequency for gene therapy.</a> Human Gene Therapy. 33 (3-4), 188-201 (2021).</li><li>Karuppusamy, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CCR5+gene+edited+CD34++CD90++hematopoietic+stem+cell+population+serves+as+an+optimal+graft+source+for+HIV+gene+therapy.">The CCR5 gene edited CD34+ CD90+ hematopoietic stem cell population serves as an optimal graft source for HIV gene therapy.</a> Frontiers in Immunology. 13, 792684 (2022).</li><li>Hopman, R. K., DiPersio, J. 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The authors declare that no competing financial interests exist.

The successful outcome of HSPC gene therapy relies predominantly on the quality and quantity of engraftable HSCs in the graft. However, the functional properties of HSCs are highly affected during the preparatory phase of gene therapy products, including by in vitro culture and toxicity associated with the gene manipulation procedure. To overcome these limitations, we have identified ideal HSPCs culture conditions that retain the stemness of CD34+CD133+CD90+ HSCs in ex&#160;v...

The inaccessibility to human leukocyte antigen (HLA)-matched donors in allogenic transplantation settings and the rapid development of highly versatile and safe genetic engineering tools make autologous hematopoietic stem cell transplantation (HSCT) a curative treatment strategy for the hereditary blood disorders1,2. Autologous hematopoietic stem and progenitor cell (HSPC) gene therapy involves the collection of patients&#39; HSPCs, genetic manipulation, correction of disease-causing mutations, and transplantation of gene-corrected HSPCs into the patient3,4. However, the successful outcome of the gene therapy relies on the quality of the transplantable gene-modified graft. The gene manipulation steps and ex vivo culture of HSPCs affect the quality of the graft by decreasing the frequency of long-term hematopoietic stem cells (LT-HSCs), necessitating the infusion of large doses of gene-manipulated HSPCs2,5,6.
Several small molecules, including SR1 and UM171, are currently being employed to expand cord blood HSPCs robustly7,8. For adult HSPCs, due to the higher cell yield obtained on mobilization, robust expansion is not required. However, retaining the stemness of isolated HSPCs in ex vivo culture is crucial for its gene therapy applications. Therefore, an approach focusing on the culture enrichment of hematopoietic stem cells (HSCs) is developed using a combination of small molecules: Resveratrol, UM729, and SR1 (RUS)7. The optimized HSPC culture conditions promote the enrichment of HSCs, resulting in increased frequency of gene-modified HSCs in vivo, and reduce the need for gene manipulating large doses of HSPCs, facilitating cost-effective gene therapy approaches8.
Here, a comprehensive protocol for HSPCs culture is described, along with the infusion and analysis of gene-edited cells in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >4D-Nucleofector&#174; X Unit</td>
			<td >LONZA BIOSCIENCE</td>
			<td >AAF-1003X</td>
			<td ></td>
		</tr>
		<tr >
			<td >4D-Nucleofector&#8482; X Kit ( 16-well Nucleocuvette&#8482; Strips)</td>
			<td >LONZA BIOSCIENCE</td>
			<td >V4XP-3032</td>
			<td></td>
		</tr>
		<tr >
			<td >Antibiotic-Antimycotic (100X)</td>
			<td >THERMO SCIENTIFIC</td>
			<td >15240096</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD45 APC</td>
			<td >BD BIOSCIENCE&#160;</td>
			<td >555485&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD13 PE</td>
			<td >BD BIOSCIENCE</td>
			<td >555394</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD19 PerCP</td>
			<td >BD BIOSCIENCE</td>
			<td >340421</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD3 PE-Cy7</td>
			<td >BD BIOSCIENCE</td>
			<td >557749</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD90 APC</td>
			<td >BD BIOSCIENCE</td>
			<td>561971</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD133/1&#160;</td>
			<td >Miltenyibiotec</td>
			<td >130-113-673</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD34 PE</td>
			<td >BD BIOSCIENCE</td>
			<td >348057</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-mouse CD45.1 PerCP-Cy5</td>
			<td >BD BIOSCIENCE</td>
			<td >560580</td>
			<td></td>
		</tr>
		<tr >
			<td >Blood Irradator-2000</td>
			<td >&#160;BRIT (Department of Biotechnology, India)</td>
			<td >BI 2000&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture dish (delta surface-treated 6-well plates)</td>
			<td >NUNC (THERMO SCIENTIFIC)</td>
			<td >140675</td>
			<td></td>
		</tr>
		<tr >
			<td >CrysoStor CS10</td>
			<td >BioLife solutions</td>
			<td >#07952</td>
			<td></td>
		</tr>
		<tr >
			<td >Busulfan</td>
			<td >CELON LABS (60mg/10mL)</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Guide-it Recombinant Cas9</td>
			<td >TAKARA BIO</td>
			<td >632640</td>
			<td></td>
		</tr>
		<tr >
			<td >Cas9-eGFP</td>
			<td >SIGMA</td>
			<td >C120040&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Centrifuge tube-15ml</td>
			<td >CORNING</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Centrifuge tube-50ml</td>
			<td >NUNC (THERMO SCIENTIFIC)</td>
			<td >339652</td>
			<td></td>
		</tr>
		<tr >
			<td >DMSO</td>
			<td >MPBIO</td>
			<td >219605590</td>
			<td></td>
		</tr>
		<tr >
			<td >DNAase</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >6469</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#8242;s Phosphate Buffered Saline- 1X</td>
			<td >HYCLONE</td>
			<td >SH30028.02</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep&#8482; Human CD34 Positive Selection Kit II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td>17856</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep magnet</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >18000</td>
			<td></td>
		</tr>
		<tr >
			<td >Electrophoresis unit</td>
			<td >ORANGE INDIA</td>
			<td >HDS0036</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >THERMO SCIENTIFIC</td>
			<td >10270106</td>
			<td></td>
		</tr>
		<tr >
			<td >Flow cytometer &#8211; ARIA III</td>
			<td >BD BIOSCIENCE</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >FlowJo&#160;</td>
			<td >BD BIOSCIENCE</td>
			<td >&#160;-</td>
			<td></td>
		</tr>
		<tr >
			<td >Flt3-L</td>
			<td >PEPROTECH</td>
			<td >300-19-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Gel imaging system</td>
			<td >CELL BIOSCIENCES</td>
			<td >11630453</td>
			<td></td>
		</tr>
		<tr >
			<td >HighPrep DTR reagent</td>
			<td >MAGBIOGENOMICS</td>
			<td >DT-70005</td>
			<td></td>
		</tr>
		<tr >
			<td >Human BD Fc Block</td>
			<td >BD BIOSCIENCE</td>
			<td >553141</td>
			<td></td>
		</tr>
		<tr >
			<td >IL6</td>
			<td >PEPROTECH</td>
			<td >200-06-50</td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM media</td>
			<td >THERMO SCIENTIFIC</td>
			<td >12440053</td>
			<td></td>
		</tr>
		<tr >
			<td >Infrared lamp</td>
			<td >MURPHY</td>
			<td >-</td>
			<td></td>
		</tr>
		<tr >
			<td >Insulin syringe 6mm 31G</td>
			<td >BD BIOSCIENCE</td>
			<td >324903</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >KETMIN 50</td>
			<td >-</td>
			<td></td>
		</tr>
		<tr >
			<td >Loading dye 6X</td>
			<td >TAKARA BIO</td>
			<td >9156</td>
			<td></td>
		</tr>
		<tr >
			<td >Lymphoprep</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >7851</td>
			<td></td>
		</tr>
		<tr >
			<td >Mice Restrainer</td>
			<td >AVANTOR</td>
			<td >TV-150</td>
			<td></td>
		</tr>
		<tr >
			<td >Nano drop spectrophotometer</td>
			<td >THERMO SCIENTIFIC</td>
			<td >ND-2000C</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer cell counting chamber</td>
			<td >ROHEM INSTRUMENTS</td>
			<td >CC-3073</td>
			<td></td>
		</tr>
		<tr >
			<td >NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)</td>
			<td >The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:005557</td>
			<td></td>
		</tr>
		<tr >
			<td >NOD,B6.SCID Il2r&#947;&#8722;/&#8722;KitW41/W41 (NBSGW)</td>
			<td >The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:026622</td>
			<td></td>
		</tr>
		<tr >
			<td >Nunc delta 6-well plate</td>
			<td >THERMO SCIENTIFIC</td>
			<td >140675</td>
			<td></td>
		</tr>
		<tr >
			<td >Polystyrene round-bottom tube</td>
			<td >BD</td>
			<td >352008</td>
			<td></td>
		</tr>
		<tr >
			<td >P3 primary cell Nucleofection solution</td>
			<td >LONZA BIOSCIENCE</td>
			<td >PBP3-02250</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipette</td>
			<td >FISHER SCIENTIFIC</td>
			<td >13-678-20A</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR clean-up kit</td>
			<td >TAKARA BIO</td>
			<td >740609.25</td>
			<td></td>
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		<tr >
			<td >Mouse Pie Cage</td>
			<td>FISCHER SCIENTIFIC</td>
			<td>50-195-5140</td>
			<td></td>
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			<td >polystyrene round-bottom tube (12 x 75 mm)</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >38007</td>
			<td></td>
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			<td >Primer3</td>
			<td >Whitehead Institute for Biomedical Research</td>
			<td >https://primer3.ut.ee/</td>
			<td></td>
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			<td >QuickExtract&#8482; DNA Extraction Solution</td>
			<td >Lucigen</td>
			<td >QE09050</td>
			<td></td>
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		<tr >
			<td >Reserveratrol</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72862</td>
			<td></td>
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			<td >SCF</td>
			<td >PEPROTECH</td>
			<td >300-07-1000</td>
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			<td >SFEM-II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >9655</td>
			<td></td>
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			<td >sgRNA</td>
			<td >SYNTHEGO</td>
			<td >-&#160;</td>
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			<td >TARSON</td>
			<td >1020</td>
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			<td >StemReginin 1</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72342</td>
			<td></td>
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			<td >ICE analysis tool</td>
			<td >SYNTHEGO</td>
			<td >https://ice.synthego.com/</td>
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			<td >Tris-EDTA buffer solution (TE) 1X</td>
			<td >SYNTHEGO</td>
			<td >Supplied with gRNA&#160;</td>
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			<td >Thermocycler</td>
			<td >APPLIED BIOSYSTEMS</td>
			<td >4375305</td>
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			<td >TPO</td>
			<td >PEPROTECH</td>
			<td >300-18-1000</td>
			<td></td>
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			<td >Trypan blue</td>
			<td >HIMEDIA LABS</td>
			<td >TCL046</td>
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			<td >UM171</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72914</td>
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			<td >Xylazine</td>
			<td >XYLAXIN - INDIAN IMMUNOLOGICALS LIMITED</td>
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crispr/cas9 gene editing of hematopoietic stem and progenitor cells for gene therapy applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present study identifies ideal HSPC culture conditions that facilitate the retention of CD34+CD133+CD90+ HSCs in ex vivo culture. To demonstrate the culture enrichment of HSCs along with the enhanced generation of gene-modified HSCs, the optimized procedures for PBMNC isolation, CD34+ cell purification, culture, gene editing, transplantation, characterization of engraftment, and gene-modified cells in vivo are provided (Figure 1</stron...

Watch this Scientific Journal Video about CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications at JoVE.com

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

crisprcas9-gene-editing-hematopoietic-stem-progenitor-cells-for-gene

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JoVE Journal

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3.3K Views.  Christian Medical College campus. The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

Video: CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

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

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

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

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

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Quantification of Viral Vectors in Tears Using ddPCR for Bioshedding Studies

A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.

Author Spotlight: Addressing Regulatory Gaps in Molecular Studies by Quantifying Viral Vectors in Complex Matrices 

Here, we present a protocol for the development and validation of good laboratory practices in the compliant detection of adeno-associated viral vectors in human tears by droplet digital polymerase chain reaction in support of clinical development of gene therapy vectors.

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. ...

Developing Gene Transformation and Editing Methods in Bamboo

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

Author Spotlight: Advancing Gene Editing in Bamboo Leaves for Sustainable Plastic Alternatives 

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...