ioGlutamatergic Neurons HTT 50CAG/WT (ab303448)
Overview
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Product name
ioGlutamatergic Neurons HTT 50CAG/WT -
Parental Cell Line
iPSC -
Organism
Human -
Tested applications
Suitable for: Next Generation Sequencing, RT-PCR, Functional Studies, ICC/IF, SDS-PAGEmore details -
Biosafety level
1 -
General notes
Wild type ioGlutamatergic Neurons (ab303447) form the genetically matched control for the ioGlutamatergic Neurons HTT50CAG/WT disease model. This physiologically-relevant isogenic pairing offers a powerful next generation model to study Huntington’s disease in research and drug discovery.
ioGlutamatergic Neurons HTT50CAG/WT are ioGlutamatergic Neurons carrying the disease-relevant 50 CAG trinucleotide repeat expansion, associated with Huntington’s disease. ioGlutamatergic Neuron HTT50CAG/WT have been reprogrammed from human iPSCs using opti-ox technology, a precise reprogramming technology. Using CRISPR/Cas9 genome editing an abnormal expansion of 50 CAG repeats has been introduced in the first exon of the Huntingtin gene. Human stem cells, within days, convert consistently into mature, functional glutamatergic neurons providing a high quality human model for the study of Huntington’s disease.
ioGlutamatergic Neurons HTT50CAG/WT express pan-neuronal and glutamatergic markers TUBB3, MAP2 and VGLUT2 by day 11, as well as the disease-relevant Huntingtin protein.
This disease model offers a fast and easy-to-use system for investigations into the impact of gene function on disease progression against an isogenic control.
In partnership with bit.bio
Karyotype: Normal
Seeding Density: 30,000 cells/cm2
Seeding compatibility: 6-, 12-, 24-, 96- and 384-well compatible
Quality control: ICC and gene expression analysis
Research applications: Academic research, Drug development, Neurotoxicology, Genetic screening (e.g. CRISPR screening).
This product is subject to limited use licenses from iPS Academia Japan Inc, TET Systems GmbH, ERS Genomics Limited and Sigma-Aldrich Co. LLC and is developed with Bit Bio patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
Properties
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Number of cells
Small: >1 x 106 cells/vial; Large: >5 x 106 cells/vial -
Viability
>85% -
Cell type
glutamatergic neuron -
Gender
Male -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituent: 10% Dimethylsulfoxide -
Research areas
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab303448 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| Next Generation Sequencing |
Use at an assay dependent concentration.
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| RT-PCR |
Use at an assay dependent concentration.
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| Functional Studies |
Use at an assay dependent concentration.
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| ICC/IF |
Use at an assay dependent concentration.
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| SDS-PAGE |
Use at an assay dependent concentration.
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| Notes |
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Next Generation Sequencing
Use at an assay dependent concentration. |
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RT-PCR
Use at an assay dependent concentration. |
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Functional Studies
Use at an assay dependent concentration. |
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ICC/IF
Use at an assay dependent concentration. |
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SDS-PAGE
Use at an assay dependent concentration. |
Images
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Immunocytochemistry/ Immunofluorescence - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)
Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of pan-neuronal proteins (MAP2 and TUBB3) in ioGlutamatergic Neurons HTT50CAG/WT compared to the isogenic control. 100X magnification.
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Immunocytochemistry/ Immunofluorescence - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of glutamatergic neuron-specific transporter (VGLUT2) in ioGlutamatergic Neurons HTT50CAG/WT compared to the isogenic control. 100X magnification.
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Functional Studies - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)ioGlutamatergic Neurons HTT50CAG/WT mature rapidly and form structural neuronal networks over 11 days, when compared to the isogenic control. Day 1 to 11 post thawing; 100X magnification.
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SDS-PAGE - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)(A) Successful on-target integration into one HTT allele confirmed by gel electrophoresis. Genotyping primers flanking the endogenous HTT CAG repeat expansion region produce a band at approximately 320bp, by PCR, in both isogenic control (ioGlutamatergic Neurons) and disease model (ioGlutamatergic Neurons HTT50CAG/WT). PCR fragments at 395bp detect on-target gene editing and introduction of a 50 CAG repeat expansion in ioGlutamatergic Neurons HTT50CAG/WT only. (B) Amplicon PCR of the plasmid donor reveals no random integration in genomic DNA from targeted colonies via gel electrophoresis. Off-target random insertion of the donor template (used to introduce the 50 CAG repeat expansion at the WT HTT locus) is detected by PCR amplification of the donor vector backbone. This is not detected in the samples from ioGlutamatergic Neurons HTT50CAG/WT.
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RT-PCR - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)Gene expression analysis demonstrates that ioGlutamatergic Neurons HTT50CAG/WT (50CAG/WT) and the isogenic control (WT) at Day 11 lack the expression of pluripotency makers (NANOG and OCT4) whilst robustly expressing pan-neuronal (TUBB3 and SYP) and glutamatergic specific (VGLUT1 and VGLUT2) markers, as well as the glutamate receptor GRIA4. Gene expression levels assessed by RT-qPCR (data expressed relative to the parental hiPSC control (iPSC Control), normalised to HMBS). Data represents Day 11 post-revival samples; n=2 biological replicates.
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RT-PCR - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)RT-qPCR analysis demonstrates similar expression level of the Huntingtin gene in both wild type ioGlutamatergic Neurons (WT) and ioGlutamatergic Neurons HTT50CAG/WT (50CAG) at day 11 post-revival (n=2 replicates). cDNA samples of the parental iPSC line
(iPSC control) were included as a reference. -
Next Generation Sequencing - ioGlutamatergic Neurons HTT 50CAG/WT (AB303448)NGS-Amplicon Sequencing was employed to confirm the number of CAG repeats in both the ioGlutamatergic Neurons HTT50CAG/WT (orange) and isogenic control (black). The number of CAG repeat reads peak at 24 for both the isogenic control and ioGlutamatergic Neurons HTT50CAG/WT. The 50 CAG repeat expansion was detected only in the ioGlutamatergic Neurons HTT50CAG/WT (orange) confirming a successful introduction of a heterozygous
50 CAG repeat expansion.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab303448 has not yet been referenced specifically in any publications.