Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

CRISPR-Cas9 based mostly knockout methods are more and more used to research gene perform. Nonetheless, redundancies and overlapping capabilities in organic signaling pathways can name for producing multi-gene knockout cells, which stays a comparatively laborious course of.
Right here we element the appliance of multi-color LentiCRISPR vectors to concurrently generate single and a number of knockouts in human cells. We offer a whole protocol, together with information RNA design, LentiCRISPR cloning, viral manufacturing and transduction, in addition to methods for sorting and screening knockout cells.
The validity of the method is demonstrated by the simultaneous deletion of as much as 4 programmed cell dying mediators in leukemic cell strains and patient-derived acute lymphoblastic leukemia xenografts, wherein single cell cloning isn’t possible.
This protocol allows any lab with entry to primary mobile biology gear, a biosafety stage 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell strains or major cells effectively inside one month.

Technology of an Akaluc knock-in human embryonic stem cell reporter line utilizing CRISPR-Cas9 expertise

Akaluc, an enzyme engineered from luciferase, offers a possible highly effective device for tracing transplanted cells in vivo due to its near-infrared emission mild.
To allow analysis of its efficiency, we inserted the Akaluc gene at AAVS1 locus utilizing CRISPR/Cas9 expertise and generated a clonal human embryonic stem steady cell line (Named H1-AAVS1-EF1α-Akaluc-KI or AkalucHES).
AkalucHES might effectively specific Akaluc and had been traced simply in vivo. We verified that AkalucHES expressed the pluripotency markers and confirmed regular stem cell morphology.
Moreover, AkalucHES maitains regular karyotype and is ready to differentiate towards three germ-layer in vivo. So the Akaluc is efficient for tracing transplanted cells in vivo.

Derivation of human pluripotent stem cell line by way of CRISPR/Cas9 mediated deletion of exon three LAMA2 gene (DMBi001-A-1)

LAMA2-related muscular dystrophy (LAMA2-MD) outcomes from mutations in LAMA2 gene, encoding laminin α-2. It’s a congenital illness characterised by muscle losing, with essentially the most extreme model being recognized inside first few months after delivery.
To generate LAMA2-DM in vitro mannequin, we excised exon three from the LAMA2 gene in our beforehand derived wholesome human induced pluripotent stem cells (hiPSCs).
Obtained hiPSCs present expression of pluripotency markers, differentiation capability into all three germ layers, regular karyotype and lack of LAMA2 expression on mRNA and protein stage after differentiation into skeletal myocytes.
Accordingly, it might present novel perception into the molecular foundation of LAMA2-MD.

Technology of an ACTA2-EGFP reporter human induced pluripotent stem cell line, KITi001-C-41, utilizing CRISPR/Cas9-mediated homologous recombination

Alpha-smooth muscle actin (α-SMA) is encoded by ACTA2 and is a key protein within the mobile contractile system of varied mesodermal cell varieties, together with hepatic stellate cells (HSCs), {smooth} muscle cells, and cardiomyocytes.
α-SMA, which is a key protein within the improvement of hepatic fibrosis, is broadly used as a dependable marker of HSC activation. Right here, we generated an ACTA2-EGFP reporter human induced pluripotent stem cell line, KITi001-C-41, utilizing a CRISPR/Cas9-based knock-in system.
These reporter hiPSC strains can be utilized for lineage tracing of mesodermal cells and for screening of HSC activation components.

Technology of a homozygous LRPAP1 knockout human embryonic stem cell line (FDCHDPe009-B) by CRISPR/Cas9 system

The homozygous autosomal recessive truncating mutations of LDL receptor associated protein related protein 1 (LRPAP1) is a attainable purpose for Nonsyndromic Excessive Myopia, sufferers with which present typical chorioretinal degeneration.
We generated an LRPAP1 knockout FDCHDPe009-B embryonic stem cell line to check mechanisms of retinal degeneration underlying LRPAP1 deficiency with the assistance of the CRISPR/Cas9 system.
Two distinct biallelic deletions within the cell line have been confirmed, which inflicting a frameshift and untimely cease codons thus affect the interpretation of LRPAP1. FDCHDPe009-B has maintained regular stem cell morphology, pluripotent gene expression, parental karyotype, and talent to distinguish into three germ layers.

Institution of a KCNQ1 homozygous knockout human embryonic stem cell line by episomal vector-based CRISPR/Cas9 system

As a member of the voltage-gated potassium ion channels, KCNQ1 performs an vital position in coronary heart physiological capabilities. Quite a few mutations in KCNQ1 had been recognized as major causes to hereditary long-QT syndrome.
To additional examine the position of KCNQ1 in human cardiac capabilities, right here we generated a homozygous KCNQ1 knockout human embryonic stem cell line (KCNQ1-KO) utilizing episomal vector-based CRISPR/Cas9 system.
This generated cell line offered typical stem cells colony morphology, maintained extremely pluripotency and regular karyotype, additionally was capable of differentiate into all three germ layers in vivo.

Deciphering the Nature of Trp 73 Isoforms in Mouse Embryonic Stem Cell Fashions: Technology of Isoform-Particular Poor Cell Traces Utilizing the CRISPR/Cas9 Gene Enhancing System

The p53 household has been broadly studied for its position in numerous physiological and pathological processes. Imbalance of p53 household proteins could contribute to developmental abnormalities and pathologies in people.
This household exerts its capabilities by way of a profusion of isoforms which are generated by totally different promoter utilization and different splicing in a cell kind dependent method. Specifically, the Trp73 gene offers rise to TA and DN-p73 isoforms that confer p73 a twin nature.
The organic relevance of p73 doesn’t solely depend on its tumor suppression results, however on its pivotal position in a number of developmental processes. Subsequently, the technology of mobile fashions that enable the examine of the person isoforms in a physiological context is of nice biomedical relevance.
We generated particular TA and DN-p73-deficient mouse embryonic stem cell strains utilizing the CRISPR/Cas9 gene enhancing system and validated them as physiological bona fide p73-isoform knockout fashions. International gene expression evaluation revealed isoform-specific alterations of distinctive transcriptional networks.
Elimination of TA or DN-p73 is suitable with pluripotency however prompts naïve pluripotent stem cell transition into the primed state, compromising ample lineage differentiation, thus suggesting that differential expression of p73 isoforms acts as a rheostat throughout early cell destiny willpower.

Genome enhancing of a hybridoma cell line by way of the CRISPR/Cas9 system: A brand new strategy for constitutive high-level expression of heterologous proteins in eukaryotic system

The ability of the CRISPR/Cas9 system has revolutionized genome enhancing in lots of fields of biology. These purposes have expanded exponentially over latest years, together with these relating to protein expression applied sciences.
The CRISPR/Cas9 system avoids random integration of the gene of curiosity and as a result of this attribute will be exploited to acquire a steady cell line for the high-yield expression of recombinant proteins. Right here we suggest a technique to edit a hybridoma cell line for the constitutive expression of proteins of curiosity utilizing the CRISPR/Cas9 system.
First, with the scope of optimizing the strategy, we changed a part of the sunshine chain of immunoglobulin with the Inexperienced Fluorescent Protein (GFP) gene, acquiring a exact knock-in within the hybridoma genome.
We confirmed the expression and secretion of GFP into the tradition medium by way of fluorimetric evaluation, in addition to appropriate genome enhancing by RNA sequencing.
Then, utilizing the identical strategy, we included the gene encoding a protein of diagnostic curiosity, the Bovine Herpesvirus 1 glycoprotein E, within the donor DNA. We obtained a steady clone capable of secrete gE protein in fusion with GFP into the tradition medium.
This consequence was confirmed by ELISA and Western Blot evaluation. This examine confirms the suitability of this cell line for the manufacturing of proteins of diagnostic curiosity by steady gene expression in a mammalian system.

NIH3T3/Cas9 Cell Line

AKR-5104 1 vial
EUR 572

293/Cas9 Cell Line

AKR-5110 1 vial
EUR 572

HeLa/Cas9 Cell Line

AKR-5111 1 vial
EUR 572

293AD Cell Line

AD-100 1 vial
EUR 461
Description: The 293AD Cell Line is derived from the parental 293 cells but selected for attributes that increase adenovirus production, including firmer attachment and larger surface area.

293AAV Cell Line

AAV-100 1 vial
EUR 508
Description: The 293AAV Cell Line is derived from the parental 293 cells but selected for attributes that increase AAV production, including firmer attachment and larger surface area.

293LTV Cell Line

LTV-100 1 vial
EUR 508
Description: The 293LTV Cell Line is derived from the parental 293 cells but selected for attributes that increase lentiviral production, including fimrer attachment and larger surface area.

293RTV Cell Line

RV-100 1 vial
EUR 508
Description: The 293RTV Cell Line is derived from the parental 293 cells but selected for attributes that increase retroviral production, including fimrer attachment and larger surface area.

293/GFP Cell Line

AKR-200 1 vial
EUR 572
Description: 293/GFP Cell Line stably expresses GFP and otherwise exhibits the same characteristics of the parental cell line.

T47D/GFP Cell Line

AKR-208 1 vial
EUR 572
Description: T47D/GFP Cell Line stably expresses GFP and otherwise exhibits the same characteristics of the parental cell line.

A549/GFP Cell Line

AKR-209 1 vial
EUR 572
Description: A549/GFP Cell Line stably expresses GFP and otherwise exhibits the same characteristics of the parental cell line.

HeLa/GFP Cell Line

AKR-213 1 vial
EUR 572
Description: HeLa/GFP Cell Line stably expresses GFP and otherwise exhibits the same characteristics of the parental cell line.

NIH3T3/GFP Cell Line

AKR-214 1 vial
EUR 572
Description: NIH3T3/GFP Cell Line stably expresses GFP and otherwise exhibits the same characteristics of the parental cell line.

SKOV-3/Luc Cell Line

AKR-232 1 vial
EUR 572
Description: SKOV-3/Luc Cell Line stably expresses luciferase and otherwise exhibits the same characteristics of the parental cell line.

MCF-7/Luc Cell Line

AKR-234 1 vial
EUR 572
Description: MCF-7/Luc Cell Line stably expresses luciferase and otherwise exhibits the same characteristics of the parental cell line.

OVCAR-5/RFP Cell Line

AKR-254 1 vial
EUR 572
Description: OVCAR-5/RFP Cell Line stably expresses RFP and otherwise exhibits the same characteristics of the parental cell line.

Platinum-E Retroviral Packaging Cell Line, Ecotropic

RV-101 1 vial
EUR 920
Description: Conventional cells used for retrovirus packaging, such as those based on NIH3T3 cells, have limited stability and produce relatively low yields of retrovirus, mainly due to the poor expression of retroviral structure proteins (gag, pol and env) in the cells. The Platinum Retroviral Packaging Cell Lines are based on the 293T cell line. They exhibit longer stability and produce higher yields of retroviral structure proteins. Plat-E cells contain gag, pol and env genes, allowing retroviral packaging with a single plasmid transfection.

Platinum-A Retroviral Packaging Cell Line, Amphotropic

RV-102 1 vial
EUR 920
Description: Conventional cells used for retrovirus packaging, such as those based on NIH3T3 cells, have limited stability and produce relatively low yields of retrovirus, mainly due to the poor expression of retroviral structure proteins (gag, pol and env) in the cells. The Platinum Retroviral Packaging Cell Lines are based on the 293T cell line. They exhibit longer stability and produce higher yields of retroviral structure proteins. Plat-A cells contain gag, pol and env genes, allowing retroviral packaging with a single plasmid transfection.

Platinum-GP Retroviral Packaging Cell Line, Pantropic

RV-103 1 vial
EUR 920
Description: Conventional cells used for retrovirus packaging, such as those based on NIH3T3 cells, have limited stability and produce relatively low yields of retrovirus, mainly due to the poor expression of retroviral structure proteins (gag, pol and env) in the cells. The Platinum Retroviral Packaging Cell Lines are based on the 293T cell line. They exhibit longer stability and produce higher yields of retroviral structure proteins. Plat-GP cells contain the gag and pol genes required for retroviral packaging; an expression vector is co-transfected with a VSVG envelope vector.

Total Protein - Murine Embryonic Stem Cell Line D3

CBA-305 500 ?g
EUR 345
Description:
  • Isolated from mouse ES-D3 cell line
  • Presented as 500 µg at 1 mg/mL in NP-40 Solubilization Buffer

Cas9 monoclonal antibody [Clone 7A9]

CAS9-AB-2 25 ug
EUR 204
  • Category: Cas9

Cas9 RT-PCR Primer Set (50ul at 5uM for each primer set)

CAS9-PR-1 50 reactions
EUR 153
  • Category: Cas9

CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric

CBA-135 96 assays
EUR 821
Description: CytoSelect 96-Well Cell Transformation Assays (Cell Recovery Compatible) provide a robust system for detecting transformed cells, screening cell transformation inhibitors, and determining in vitro drug sensitivity. A proprietary modified soft agar matrix allows you to either quantify cells using the included fluorescent dye, or recover the cells for further analysis.

CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric

CBA-135-5 5 x 96 assays
EUR 3356
Description: CytoSelect 96-Well Cell Transformation Assays (Cell Recovery Compatible) provide a robust system for detecting transformed cells, screening cell transformation inhibitors, and determining in vitro drug sensitivity. A proprietary modified soft agar matrix allows you to either quantify cells using the included fluorescent dye, or recover the cells for further analysis.

CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Fluorometric

CBA-140 96 assays
EUR 856
Description: CytoSelect 96-Well Cell Transformation Assays (Cell Recovery Compatible) provide a robust system for detecting transformed cells, screening cell transformation inhibitors, and determining in vitro drug sensitivity. A proprietary modified soft agar matrix allows you to either quantify cells using the included fluorescent dye, or recover the cells for further analysis.

CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Fluorometric

CBA-140-5 5 x 96 assays
EUR 3483
Description: CytoSelect 96-Well Cell Transformation Assays (Cell Recovery Compatible) provide a robust system for detecting transformed cells, screening cell transformation inhibitors, and determining in vitro drug sensitivity. A proprietary modified soft agar matrix allows you to either quantify cells using the included fluorescent dye, or recover the cells for further analysis.

StemTAG Stem Cell Colony Formation Assay (Cell Recovery Compatible)

CBA-325 96 assays
EUR 856
Description: Our StemTAG 96-Well Stem Cell Colony Formation Assay provides a high-throughput method to quantify ES cells in just 7-10 days, and no manual cell counting is required. Once colonies are formed, they may be analyzed in three different ways: 1. Lyse cells, then quantify in a fluorescence plate reader using dye included in the kit; 2. Lyse cells, then quantify alkaline phosphatase activity using reagents provided; or 3. Recover colonies from matrix for further culture or analysis.

StemTAG Stem Cell Colony Formation Assay (Cell Recovery Compatible)

CBA-325-5 5 x 96 assays
EUR 3361
Description: Our StemTAG 96-Well Stem Cell Colony Formation Assay provides a high-throughput method to quantify ES cells in just 7-10 days, and no manual cell counting is required. Once colonies are formed, they may be analyzed in three different ways: 1. Lyse cells, then quantify in a fluorescence plate reader using dye included in the kit; 2. Lyse cells, then quantify alkaline phosphatase activity using reagents provided; or 3. Recover colonies from matrix for further culture or analysis.

CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric, Trial Size

CBA-135-T 24 assays
EUR 432
Description: CytoSelect 96-Well Cell Transformation Assays (Cell Recovery Compatible) provide a robust system for detecting transformed cells, screening cell transformation inhibitors, and determining in vitro drug sensitivity. A proprietary modified soft agar matrix allows you to either quantify cells using the included fluorescent dye, or recover the cells for further analysis.

CytoSelect 96-Well Cell Transformation Assay (Cell Recovery Compatible, Fluorometric), Trial Size

CBA-140-T 24 assays
EUR 456
Description: CytoSelect 96-Well Cell Transformation Assays (Cell Recovery Compatible) provide a robust system for detecting transformed cells, screening cell transformation inhibitors, and determining in vitro drug sensitivity. A proprietary modified soft agar matrix allows you to either quantify cells using the included fluorescent dye, or recover the cells for further analysis.

Collagen-based Cell Contraction Assay

CBA-201 24 assays
EUR 485
Description: Cell Biolabs? Collagen-based Contraction Assay Kit provides a simple system to assess cell contractivity in vitro and screen cell contraction mediators. Each kit provides sufficient quantities to perform up to 24 assays in a 24-well plate. The kit can be also used in culturing cells in 3D collagen matrix.

CytoSelect MTT Cell Proliferation Assay

CBA-252 960 assays
EUR 409
Description: Cell Biolabs? CytoSelect MTT Cell Proliferation Assay provides a colorimetric format for measuring and monitoring cell proliferation.  The kit contains sufficient reagents for the evaluation of 960 assays in 96-well plates or 192 assays in 24-well plates.  Cells can be plated and then treated with compounds or agents that affect proliferation.  Cells are then detected with the proliferation reagent, which is converted in live cells from the yellow tetrazole MTT to the purple formazan form by a cellular reductase (Figure 1).  An increase in cell proliferation is accompanied by an increased signal, while a decrease in cell proliferation (and signal) can indicate the toxic effects of compounds or suboptimal culture conditions.  The assay principles are basic and can be applied to most eukaryotic cell lines, including adherent and non-adherent cells and certain tissues.  This cell proliferation reagent can be used to detect proliferation in bacteria, yeast, fungi, protozoa as well as cultured mammalian and piscine cells.

CytoSelect 24-well Cell Invasion, Fluorometric

CBA-111 12 assays
EUR 595
Description: The ability of malignant tumor cells to invade normal surrounding tissue contributes in large part to the morbidity and mortality of cancers. Cell invasion requires several distinct cellular functions including adhesion, motility, detachment, and extracellular matrix proteolysis. Our CytoSelect Cell Invasion Assays utilize precoated inserts to assay the invasive properties of tumor cells. Invasive cells can be quantified in 24-well plates on either a standard microplate reader or a fluorescence plate reader. Inserts are precoated on the top of the membrane with ECM matrix gel (basement membrane), a protein mix isolated from EHS tumor cells.

CytoSelect 96-well Cell Invasion, Fluorometric

CBA-112 96 assays
EUR 757
Description: The ability of malignant tumor cells to invade normal surrounding tissue contributes in large part to the morbidity and mortality of cancers. Cell invasion requires several distinct cellular functions including adhesion, motility, detachment, and extracellular matrix proteolysis. Our CytoSelect 96-Well Cell Invasion Assays utilize precoated inserts to assay the invasive properties of tumor cells. Invasive cells can be quantified in 96-well plates on a fluorescence plate reader. Inserts are precoated on the top of the membrane with Basement Membrane, an ECM protein mix isolated from EHS tumor cells.

Radius 24-Well Cell Migration Assay

CBA-125 24 assays
EUR 502
Description: The Radius Cell Migration Assay provides a unique alternative to conventional cell migration assays using the Boyden chamber. Unlike Boyden chamber assays which may only be analyzed at endpoint, the Radius assay uses a proprietary cell culture plate containing a carefully-defined biocompatible hydrogel (Radius gel) spot centralized at the bottom of each well. When cells are seeded in the well, they will attach everywhere except on the Radius gel, creating a cell-free zone. Following cell seeding the Radius gel is removed, allowing migratory cells to move across the area and close the gap.

Radius 24-Well Cell Migration Assay

CBA-125-5 5 x 24 assays
EUR 1969
Description: The Radius Cell Migration Assay provides a unique alternative to conventional cell migration assays using the Boyden chamber. Unlike Boyden chamber assays which may only be analyzed at endpoint, the Radius assay uses a proprietary cell culture plate containing a carefully-defined biocompatible hydrogel (Radius gel) spot centralized at the bottom of each well. When cells are seeded in the well, they will attach everywhere except on the Radius gel, creating a cell-free zone. Following cell seeding the Radius gel is removed, allowing migratory cells to move across the area and close the gap.

Radius 96-Well Cell Migration Assay

CBA-126 96 assays
EUR 572
Description: The Radius Cell Migration Assay provides a unique alternative to conventional cell migration assays using the Boyden chamber. Unlike Boyden chamber assays which may only be analyzed at endpoint, the Radius assay uses a proprietary cell culture plate containing a carefully-defined biocompatible hydrogel (Radius gel) spot centralized at the bottom of each well. When cells are seeded in the well, they will attach everywhere except on the Radius gel, creating a cell-free zone. Following cell seeding the Radius gel is removed, allowing migratory cells to move across the area and close the gap.

Radius 96-Well Cell Migration Assay

CBA-126-5 5 x 96 assays
EUR 2248
Description: The Radius Cell Migration Assay provides a unique alternative to conventional cell migration assays using the Boyden chamber. Unlike Boyden chamber assays which may only be analyzed at endpoint, the Radius assay uses a proprietary cell culture plate containing a carefully-defined biocompatible hydrogel (Radius gel) spot centralized at the bottom of each well. When cells are seeded in the well, they will attach everywhere except on the Radius gel, creating a cell-free zone. Following cell seeding the Radius gel is removed, allowing migratory cells to move across the area and close the gap.

Radius 384-Well Cell Migration Assay

CBA-127 384 assays
EUR 601
Description: The Radius Cell Migration Assay provides a unique alternative to conventional cell migration assays using the Boyden chamber. Unlike Boyden chamber assays which may only be analyzed at endpoint, the Radius assay uses a proprietary cell culture plate containing a carefully-defined biocompatible hydrogel (Radius gel) spot centralized at the bottom of each well. When cells are seeded in the well, they will attach everywhere except on the Radius gel, creating a cell-free zone. Following cell seeding the Radius gel is removed, allowing migratory cells to move across the area and close the gap.
These experiments will allow the method to be developed from its proof of idea to extra particular purposes within the discipline of infectious illness diagnostics.

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