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.
T47D/GFP Cell Line |
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AKR-208 | Cell Biolabs | 1 vial | EUR 686.4 |
Description: T47D/GFP Cell Line stably expresses GFP and otherwise exhibits the same characteristics of the parental cell line. |
A549/GFP Cell Line |
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AKR-209 | Cell Biolabs | 1 vial | EUR 388 |
Description: Fluorescence |
HeLa/GFP Cell Line |
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AKR-213 | Cell Biolabs | 1 vial | EUR 388 |
Description: Fluorescence |
NIH3T3/GFP Cell Line |
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AKR-214 | Cell Biolabs | 1 vial | EUR 388 |
Description: Fluorescence |
MCF-7/Luc Cell Line |
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AKR-234 | Cell Biolabs | 1 vial | EUR 686.4 |
Description: MCF-7/Luc Cell Line stably expresses luciferase and otherwise exhibits the same characteristics of the parental cell line. |
SKOV-3/Luc Cell Line |
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AKR-232 | Cell Biolabs | 1 vial | EUR 686.4 |
Description: SKOV-3/Luc Cell Line stably expresses luciferase and otherwise exhibits the same characteristics of the parental cell line. |
OVCAR-5/RFP Cell Line |
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AKR-254 | Cell Biolabs | 1 vial | EUR 686.4 |
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 |
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RV-101 | Cell Biolabs | 1 vial | EUR 648 |
Platinum-GP Retroviral Packaging Cell Line, Pantropic |
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RV-103 | Cell Biolabs | 1 vial | EUR 648 |
Platinum-A Retroviral Packaging Cell Line, Amphotropic |
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RV-102 | Cell Biolabs | 1 vial | EUR 648 |
Total Protein - Murine Embryonic Stem Cell Line D3 |
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CBA-305 | Cell Biolabs | 500 ?g | EUR 414 |
Description:
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Cas9 (CRISPR Associated Protein 9) ELISA Kit |
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PRB-5079 | Cell Biolabs | 96 assays | EUR 408 |
Description: Excepted Quantities |
StemTAG Stem Cell Colony Formation Assay (Cell Recovery Compatible) |
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CBA-325 | Cell Biolabs | 96 assays | EUR 1027.2 |
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) |
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CBA-325-5 | Cell Biolabs | 5 x 96 assays | EUR 4033.2 |
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 |
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CBA-135 | Cell Biolabs | 96 assays | EUR 596 |
CytoSelect™ Cell Transformation Assay (Cell Recovery Compatible), Colorimetric |
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CBA-135-5 | Cell Biolabs | 5 x 96 assays | EUR 2592 |
CytoSelect™ Cell Transformation Assay (Cell Recovery Compatible), Fluorometric |
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CBA-140 | Cell Biolabs | 96 assays | EUR 624 |
CytoSelect™ Cell Transformation Assay (Cell Recovery Compatible), Fluorometric |
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CBA-140-5 | Cell Biolabs | 5 x 96 assays | EUR 2700 |
CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric, Trial Size |
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CBA-135-T | Cell Biolabs | 24 assays | EUR 518.4 |
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 |
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CBA-140-T | Cell Biolabs | 24 assays | EUR 547.2 |
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 |
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CBA-201 | Cell Biolabs | 24 assays | EUR 348 |
Radius 384-Well Cell Migration Assay |
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CBA-127 | Cell Biolabs | 384 assays | EUR 721.2 |
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 |
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CBA-127-5 | Cell Biolabs | 5 x 384 wells | EUR 2802 |
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. |
HIF-1 Alpha Cell Based ELISA Kit |
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CBA-281 | Cell Biolabs | 96 assays | EUR 734.4 |
Description: Cell Biolabs? HIF-1 Cell Based ELISA Kit is an immunoassay developed for rapid detection of HIF-1 Alpha in fixed cells. Cells on a microplate are stimulated for HIF-1 Alpha stabilization, fixed, permeabilized, and then neutralized in the well. HIF-1 Alpha is then detected with an anti-HIF-1 alpha antibody followed by an HRP conjugated secondary antibody. Each kit provides sufficient reagents to perform up to a total of 96 assays and can detect HIF-1 Alpha from human, mouse, or rat. |
CytoSelect™ MTT Cell Proliferation Assay |
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CBA-252 | Cell Biolabs | 960 assays | EUR 268 |
Radius™ 24-Well Cell Migration Assay |
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CBA-125 | Cell Biolabs | 24 assays | EUR 356 |
Description: Excepted Quantities |
Radius™ 24-Well Cell Migration Assay |
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CBA-125-5 | Cell Biolabs | 5 x 24 assays | EUR 1516 |
Description: Excepted Quantities |
Radius™ 96-Well Cell Migration Assay |
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CBA-126 | Cell Biolabs | 96 assays | EUR 416 |
Description: Excepted Quantities |
Radius™ 96-Well Cell Migration Assay |
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CBA-126-5 | Cell Biolabs | 5 x 96 assays | EUR 1788 |
Description: Excepted Quantities |
Radius™ 48-Well Cell Migration Assay |
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CBA-5037 | Cell Biolabs | 48 assays | EUR 372 |
Description: Excepted Quantities |
Radius™ 48-Well Cell Migration Assay |
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CBA-5037-5 | Cell Biolabs | 5 x 48 assays | EUR 1592 |
Description: Excepted Quantities |
CytoSelect Cell Proliferation Assay Reagent (Fluorometric) |
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CBA-250 | Cell Biolabs | 10 mL | EUR 490.8 |
Description: Cell Biolabs? CytoSelect Cell Proliferation Assay Reagent (Fluorometric) provides a fluorometric format for measuring and monitoring cell proliferation. Cells can be plated and then treated with compounds or agents that affect proliferation. Cells are then incubated with the proliferation reagent. Upon entering metabolically active live cells, the non-fluorescent proliferation reagent is converted into a bright red fluorescent form. An increase in cell proliferation is accompanied by increased fluorescent 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. The kit contains sufficient reagents for the evaluation of 960 assays in ten 96-well plates or 192 assays in eight 24-well plates. |
CytoSelect™ BrdU Cell Proliferation ELISA Kit |
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CBA-251 | Cell Biolabs | 96 assays | EUR 380 |
Description: Excepted Quantities |
CytoSelect™ 96-well Cell Transformation Assay |
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CBA-130 | Cell Biolabs | 96 assays | EUR 532 |
CytoSelect™ 96-well Cell Transformation Assay |
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CBA-130-5 | Cell Biolabs | 5 x 96 assays | EUR 2280 |
CytoSelect™ Cell Viability and Cytotoxicity Assay |
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CBA-240 | Cell Biolabs | 96 assays | EUR 256 |
CytoSelect 24-well Laminin Cell Invasion, Colorimetric |
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CBA-110-LN | Cell Biolabs | 12 assays | EUR 714 |
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 Laminin. |
CytoSelect™ 24-Well Cell Co-Culture System |
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CBA-160 | Cell Biolabs | 24 assays | EUR 308 |
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.