Name: genome engineering of primary human b cells using crispr/cas9
Uploaded: 2020-11-03T14:00:00
Description: Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Este trabajo fue financiado por el Children's Cancer Research Fund (CCRF) y nih R01 AI146009 a B.S.M.

Se obtuvieron muestras de leucoféresis de donantes sanos de un banco de sangre local. Todos los experimentos descritos aquí fueron determinados como exentos para la investigación por la Junta de Revisión Institucional (IRB) y fueron aprobados por el Comité Institucional de Bioseguridad (IBC) de la Universidad de Minnesota.
NOTA: Todos los experimentos se realizaron en cumplimiento de la precaución universal para patógenos transmitidos por la sangre, con técnicas estériles/asépticas y...

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M., 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=Engineering+human+hematopoietic+stem/progenitor+cells+to+produce+a+broadly+neutralizing+anti-HIV+antibody+after+in+vitro+maturation+to+human+B+lymphocytes.">Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes.</a> Blood. 113 (7), 1422-1431 (2008).</li><li>Mock, U., Thiele, R., Uhde, A., Fehse, B., Horn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+lentiviral+transduction+and+transgene+expression+in+primary+human+B+cells.">Efficient lentiviral transduction and transgene expression in primary human B cells.</a> Human Gene Therapy Methods. 23 (6), 408-415 (2012).</li><li>Heltemes-Harris, L. 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La ingeniería precisa del genoma en células B humanas primarias ha sido un reto hasta hace poco9,10. Anteriormente habíamos publicado protocolos utilizando CRISPR/Cas9 para diseñar células B humanas primarias9. Aquí, delineamos protocolos mejorados para el aislamiento, expansión e ingeniería de células B para permitir un KO eficiente de CD19 o para la EGFP.
Aquí demostramos que nuestro protoco...

Las células B son un subgrupo del linfanato derivado de células madre hematopoyéticas. Desempeñan un papel crítico en el sistema inmune humorístico adaptativo mediante la producción de grandes cantidades de anticuerpos en respuesta a los desafíos inmunes1. Las células B también son precursoras de las células B de la memoria y las células plasmáticas de larga duración diferenciadas terminalmente, proporcionando así inmunidad humorística duradera2. Las células plasmáticas, en particular, son únicas entre las células inmunes en su capacidad para producir grandes cantidades de un anticuerpo específico mientras sobreviven durante años o décadas3. Además, la facilidad de aislamiento de la sangre periférica hace que el linaje de células B sea un excelente candidato para nuevas terapias basadas en células4.
Anteriormente, los métodos de integración aleatoria, como los que utilizan vectores lentivirales o un transposon de la Bella Durmiente, se han utilizado para diseñar células B para la entrega de transgéneros y la expresión5,6,7,8. Sin embargo, la naturaleza no específica de estos enfoques dificulta el estudio de las funciones biológicas de un gen específico en las células B y conlleva un riesgo inherente de mutagénesis insertal y expresión y/o silenciamiento variable de transgénero en el entorno terapéutico.
El sistema CRISPR/Cas9 es una poderosa herramienta de ingeniería del genoma que permite a los investigadores editar con precisión el genoma de varias células en numerosas especies. Recientemente, dos grupos, incluido el nuestro, han desarrollado con éxito métodos para la expansión ex vivo y la ingeniería genómica dirigida de las células B humanas primarias9,10. Describiremos el proceso de purificación de las células B humanas primarias de una muestra de leucoforesis. Después de eso, describiremos nuestro protocolo actualizado para la expansión de células B y la activación de celdas B aisladas. A continuación, describiremos un proceso para noquear un cúmulo de diferenciación 19 (CD19), un receptor específico de células B y un sello distintivo de las células B, mediante la electroporación para introducir el ARNM CRISPR/Cas9 junto con el ARGN CD19 en las células B activadas.
Cas9 mRNA se traduce y se une al SgRNA CD19 para formar un complejo crispr/cas9-sgRNA ribonucleoprotein (RNP). Posteriormente, el esgRNA en el complejo lleva a Cas9 a crear rotura de doble hebra (DSB) en la secuencia objetivo en el exón 2 del gen. Las células repararán el OSD mediante "unión final no homóloga" mediante la introducción o eliminación de nucleótidos, lo que conduce a la mutación del cambio de trama y hace que el gen sea noqueado. A continuación, mediremos la pérdida de CD19 por citometría de flujo y analizaremos la formación de indel mediante el seguimiento de los indels mediante el análisis de la descomposición (TIDE).
A continuación, describiremos el proceso de uso de CRISPR/Cas9 junto con un vector AAV6 recombinante (rAAV6, una plantilla de donante para la reparación dirigida por homología (HDR)) para mediar la inserción específica del sitio de proteína fluorescente verde mejorada(EGFP) en el gen del sitio de integración del virus asociado al adeno 1 (AAVS1). El gen AAVS1 es un locus activo sin funciones biológicas conocidas y un sitio de integración viral AAV en el genoma humano; por lo tanto, se considera un "puerto seguro" para la ingeniería del genoma. Aquí, informamos que la expansión y activación de las células B permitió hasta 44 veces la expansión en 7 días de cultivo (Figura 1). La electroporación de las células B mostró una ligera reducción de la salud celular general(Figura 2A)a las 24 h después de la transfección. El análisis de la gráfica de dispersión del marcador CD19 (Figura 2B) mostró una reducción de hasta el 83% en las celdas editadas (Figura 2C).
El análisis tide de los cromatógrafos (Figura 3A) reveló que el % de indels era similar al % de pérdida de proteína por citometría de flujo (Figura 3B). El análisis de citometría de flujo del experimento KI mostró que las células que recibieron el vector AAV(Figura 4),junto con RNP, expresaron hasta un 64% de células positivas por EGFP(Figura 5A)y más tarde mostraron una integración exitosa por reacción en cadena de la polimerasa de unión (PCR) (Figura 5B). Los recuentos de células mostraron que todas las muestras se recuperaron rápidamente dentro de los 3 días posteriores a la ingeniería(Figura 5C).

<table>
	<tbody>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3 protein, 500 ug</td>
			<td>IDT</td>
			<td>1081059</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>Amaxa P3 primary cell 4D- Nucleofector X Kit S (32 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium-chloride-potassium (ACK) lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap chemically modified Cas9 mRNA</td>
			<td>Trilink Biotechnology</td>
			<td>L-7206-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG ODN 2006 (ODN 7909) 5 mg</td>
			<td>Invivogen</td>
			<td>TLRL-2006-5</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Cryostor CS10, 100 mL</td>
			<td>STEMCELL Technology</td>
			<td>7930</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS Immune Cell SR</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2596101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep human B cells isolation kit</td>
			<td>STEMCELL Technology</td>
			<td>17954</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience fixable viability dye eFlour 780</td>
			<td>eBiosciences</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Excellerate B cell media, Xeno-free</td>
			<td>R&#38;D Systems</td>
			<td>CCM031</td>
			<td>B-cell basal medium</td>
		</tr>
		<tr>
			<td>Falcon 14 mL Polypropylene Round-bottom Tube</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td>For thawing B cells only</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneMate SnapStrip&#174; 8-Strip 0.2 mL PCR Tubes with Individual Attached Dome Caps</td>
			<td>BioExpress</td>
			<td>T-3035-1 / 490003-692</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone 0.0067M PBS (No Ca, No Mg) or 1x PBS</td>
			<td>GE lifesciences</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza 4D Nucleofector X unit</td>
			<td>Lonza</td>
			<td>AAF-1002X</td>
			<td></td>
		</tr>
		<tr>
			<td>Mega CD40 Ligand</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-522-110-C010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Sigma-Aldrich</td>
			<td>C1562-1EA</td>
			<td>For freezing cells</td>
		</tr>
		<tr>
			<td>Pen/Strep 100X</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-AB2-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-human CD19 clone HIB19</td>
			<td>biolegend</td>
			<td>302228</td>
			<td>smaller size is also available</td>
		</tr>
		<tr>
			<td>rAAV6 SA-GFP pakaging ( with our SA-GFP cassette see Figure 4.)</td>
			<td>Vigene Biosciences</td>
			<td>N/A</td>
			<td>large scale packaging, 1e13 GC/mL, 500 mL</td>
		</tr>
		<tr>
			<td>Recombinant human IL-10 protein 250 ug</td>
			<td>R&#38;D Systems</td>
			<td>217-IL-250</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Recombinant human IL-15 protein 25 ug</td>
			<td>R&#38;D Systems</td>
			<td>247-ILB-025</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>The Big Easy EasySep Magnet</td>
			<td>STEMCELL Technology</td>
			<td>18001</td>
			<td>different sizes available</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td>Fisher Scientific</td>
			<td>BP2476100</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome engineering of primary human b cells using crispr/cas9

Las células B son linfocitos derivados de células madre hematopoyéticas y son un componente clave del brazo humorístico del sistema inmunitario adaptativo. Hacen candidatos atractivos para terapias basadas en células debido a su facilidad de aislamiento de la sangre periférica, su capacidad para expandirse in vitro, y su longevidad in vivo. Además, su función biológica normal — para producir grandes cantidades de anticuerpos — se puede utilizar para expresar cantidades muy grandes de una proteína terapéutica, como un anticuerpo recombinante para combatir la infección, o una enzima para el tratamiento de las enzimopatías. Aquí, proporcionamos métodos detallados para aislar las células B humanas primarias de células mononucleares de sangre periférica (PBMCs) y activar/expandir células B aisladas in vitro. A continuación, demostramos los pasos implicados en el uso del sistema CRISPR/Cas9 para el KO específico del sitio de genes endógenos en células B. Este método permite el KO eficiente de varios genes, que se pueden utilizar para estudiar las funciones biológicas de los genes de interés. A continuación, demostramos los pasos para utilizar el sistema CRISPR/Cas9 junto con un vector viral (rAAV) recombinante, asociado a adeno, para una integración eficiente específica del sitio de un cassette de expresión de transgénero en celdas B. Juntos, este protocolo proporciona una plataforma de ingeniería paso a paso que se puede utilizar en células B humanas primarias para estudiar las funciones biológicas de los genes, así como para el desarrollo de la terapia de células B.

El protocolo actualizado de expansión y activación permitió la rápida expansión de las células B hasta 44 veces en 7 días(Figura 1; n =3 donantes). En el experimento KO, el recuento de células B utilizando tinción azul trypan mostró más del 80% de células viables con una ligera reducción en la recuperación celular tanto en el control como en las muestras de KO CD19 a 24 h después de la electroporación(Figura 2A;p ≥ 0.05, n = 3 donantes). Este r...

Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Aquí proporcionamos un protocolo detallado paso a paso para la ingeniería genómica basada en CRISPR/Cas9 de células B humanas primarias para el knockout genético (KO) y el knock-in (KI) para estudiar las funciones biológicas de los genes en las células B y el desarrollo de la terapia de células B.

Ingeniería genómica de células B humanas primarias usando CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

This protocol allows researchers to precisely eliminate a gene in B cells to study the loss of function effects of that gene or to insert a trans gene for stable trans gene expression that can be used for research or therapeutic purposes. The main advantage of this method is that it provides a universal platform that makes it possible to easily and precisely engineer B cells with high efficiencies. This method can be used to engineer B cells to express recombinant antibodies or enzymes and then transfer those B cells to treat infections or in enzymopathies.To begin, thaw B cells in a 37 degree Celsius water bath. While waiting, transfer two milliliters of pre warmed FBS into a sterile, 15-milliliter conical tube. Once the B cells are completely thawed, immediately add one milliliter of pre-warmed FBS, drop-wise, into the sample.Incubate at room temperature for one minute. Gently pipette to resuspend the sample and transfer the whole volume, drop-wise, into a conical tube containing two milliliters of prewarmed FBS. Bring the volume to 15 milliliters with sterile PBS, then cap the tube and invert it gently two to three times.Centrifuge the sample at 400 times G for five minutes, then discard the supernatant without disturbing the cell pellet. Resuspend the pellet with one milliliter of pre-equilibrated B-cell expansion medium and count the cells. The total cell number should be approximately 10 to the seventh cells.Transfer the cells into a flask containing 20 milliliters of the pre-equilibrated B-cell expansion medium. The final concentration of the cells, should be approximately five times 10 to the fifth cells per milliliter. Incubate the flask vertically in a tissue culture incubator.Prepare the CRISPR/Cas9 transfecting substrate by mixing one microgram of chemically modified sgRNA with 1.5 micrograms of chemically modified streptococcus pyogenes Cas9 nucleus mRNA per transfection reaction. For control, add one microliter of TE buffer, instead of sgRNA, into a 0.2-milliliter tube of an eight tube strip. Turn on an electroporator and prepare the nuclear infection reagents.Count and transfer 1 million B cells per transfection reaction, into a sterile conical tube. Bring the volume to 15 milliliters with sterile PBS and centrifuge at 400 times G for five minutes. After centrifugation, discard the supernatant.Resuspend the cells with 10 milliliters of sterile PBS and centrifuge at 400 times G for five minutes, then discard the supernatant completely without disturbing the cell pellet. Add 0.5 micrograms of chemically modified GFP mRNA per 1 million B cells to the cell pellet. Resuspend the pellet with 20 microliters of primary cell transfection reagent per 1 million B cells and mix gently by pipetting five to six times.Transfer 20.5 microliters per transfection reaction into the 0.2-milliliter tubes containing CRISPR/Cas9 reagent. Pipette up and down once to mix and transfer the entire volume into a transfection cuvette. Cap and tap the cuvette on the bench gently to ensure that the liquid covers the bottom of the cuvette.Use the human primary B-cell protocol on the electroporator for transfection. Rest the electroporated cells in the cuvette at room temperature for 15 minutes. Then transfer 80 microliters of the pre-equilibrated B-cell expansion medium into the transfection reaction in the cuvette.Place the cuvette in the tissue culture incubator for 30 minutes. Gently pipe head a couple of times to mix and transfer the whole volume of the sample from the cuvette to an appropriate well of a 48-well tissue culture plate containing one milliliter of the B-cell expansion medium. The final concentration of the cells should be 1 million cells per milliliter.Performing a gene knock-in experiment, transfer recombinant adeno associated type-six viral vector at 500, 000 multiplicity of infection into the appropriate well containing electroporated cells. Place the plate in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide with humidity. In the knockout experiment, the B-cell count showed more than 80%viable cells with a slight reduction in cell recovery in both the control and the CD19 knockout samples at 24 hours post electroporation.B cells were collected on day five post-transfection for flow cytometry and tide analysis. Representative scatterplots of the control and knockout samples showed 14 and 95%CD19 negative cells respectively, demonstrating a significant reduction in CD19 expression in the knockout samples. Chromatograms of genomic sequencing showed double peaks in the CD19 knockout B cells, indicating insertions or deletions of nucleotides post-CRISPR/Cas9 mediated double-stranded breaks.Indel analysis of the chromatographs of the knockout samples showed a high percent of indel formation at the CD19 locus, which is consistent with percent CD19 protein loss detected by flow cytometry. B cells from the knock-in experiment were collected on day 12, post engineering. Scatterplots showed 64%of EGFP positive cells in the sample that received the recombinant adeno associated type-six viral vector, together with RNP, whereas none or minimal EGFP positivity was observed in the control and vector-only samples respectively.A junction PCR amplification, showed 1.5 kilobase pair amplicons in the knock-in sample and no PCR product was observed in either the control or vector-only sample. Cell counts showed that the engineering process affects cell recovery in the knock-in sample more than the control or the vector-only samples. However, all samples quickly rebounded within three days after engineering.This procedure can also be used to introduce base editor agents to alter single base pairs in order to induce or correct single point mutations in the B-cell genome. It could also be used to introduce premature stop codons or to alter splice sites, allowing for multiple gene knockouts without the risk of chromosomal translocations. Before this technique was developed, engineering human B cells would inquire the use of other more challenging gene engineering approaches.This technique is an easier, less expensive and quicker alternative. It could also potentially be used to synthesize cell therapy products for clinical use.

genome-engineering-of-primary-human-b-cells-using-crisprcas9

Research

JoVE Journal

Biology

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

La disección fina y gruesa de los epitelios sensoriales del oído interno en adultos de pez cebra ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Investigación

Biología

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Del genoma del ratón Ingeniería Utilizando Nucleasas Designer

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Generación de deleciones genómicas en líneas celulares de mamíferos a través de CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Electrofisiología de células enteras de células enterocromafines murino cultivadas primario

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

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.

Silenciar la chispa: Edición del genoma CRISPR/Cas9 en peces débilmente eléctricos

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

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Preparación e inyección de embriones de mosquitos Culex para generar mutaciones nulas utilizando CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

Construcción de mutantes homocigotos de langostas migratorias utilizando tecnología CRISPR/Cas9

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

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