hiPSC-ARX-T2A-H2B-CFP-Flag
HMGUi001-A-4
General
Donor Information
General Donor Information |
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| Sex | female |
| Ethnicity | Caucasian |
Phenotype and Disease related information (Donor) |
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| Diseases | No disease was diagnosed.
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| Disease associated phenotypes | no phenotypes |
| Family history | no |
| Is the medical history available upon request? | no |
| Is clinical information available? | no |
Karyotyping (Donor) |
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| Has the donor karyotype been analysed? |
No
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Other Genotyping (Donor) |
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| Is there genome-wide genotyping or functional data available? |
No
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Donor Relations |
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| Other cell lines of this donor | |
External Databases (Donor) |
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| BioSamples | SAMEA5696688 |
Ethics
Also have a look at the ethics information for the parental line
HMGUi001-A
.
| Is there an MTA available for the cell line? | No |
| For generation of the cell line, who was the supplier of any recombined DNA vectors or commercial kits used? | The targeting vector was generated at IDR, and the containing T2A-Sequence, H2B or CFP sequence according to IDR´s knowledge do NOT underly any constraints. |
| Are you aware of any constraints on the use or distribution of the cell line from the owner or any parties identified in the query above? | Yes |
| Constraints for use or distribution | According to donor consent the line must not be used for commercial purposes. For reserach purposes project specific review by an Ethics Committee is obligatory. |
hIPSC Derivation
General |
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The source cell information can be found in the parental cell line
HMGUi001-A.
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| Passage number reprogrammed | 25 |
Reprogramming method |
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| Vector type | Integrating |
| Vector | The Arx locus was targeted by homologous recombination and CRISPR/Cas9 technology using Arx-2A-H2B-CFP-Flag targeting vector: We cloned the genomic sequence of the ARX gene into a targeting vector and utilize the 737 bp upstream region and the 1076 bp downstream region of the translational stop codon as 5´ and 3´ homology arms (HA). Before the stop codon, we introduced a 2A sequence followed by a Histone 2B (H2B) fused to the cyan fluorescent protein (CFP) tailed by a Flag tag. Thereby the reporter line would translate equal amounts of ARX and H2B-CFP-Flag protein, separated by an autonomous intra-ribosomal self-processing of the 2A-peptide from thosea asigna virus. A gRNA, that introduced dsDNA breaks 3 bp upstream of the stop codon of the ARX was cloned into the pU6-(BbsI)sgRNA_CAG-Cas9-venus-bpA vector that allowed FACS sorting. |
| Is the used vector excisable? |
Unknown |
| Absence of reprogramming vector(s)? |
No |
| Reprogramming vectors silenced? |
No |
Vector free reprogramming |
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Other |
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| Selection criteria for clones | Cas9-Venus expression, FACS sorting, colony picking from single cell seeding |
| Derived under xeno-free conditions |
Yes |
| Derived under GMP? |
No |
| Available as clinical grade? |
No |
Culture Conditions
| Surface coating | Matrigel/Geltrex |
| Feeder cells |
No |
| Passage method |
Enzymatically
Accutase
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| O2 Concentration | 21 % |
| CO2 Concentration | 5 % |
| Medium |
Other medium:
Base medium: StemMACS iPS-Brew XF
Main protein source: xeno-free Serum concentration: 0 % |
| Has Rock inhibitor (Y27632) been used at passage previously with this cell line? | Yes |
| Has Rock inhibitor (Y27632) been used at cryo previously with this cell line? | No |
| Has Rock inhibitor (Y27632) been used at thaw previously with this cell line? | No |
Characterisation
Analysis of Undifferentiated Cells
Immunostaining showing SOX2 and OCT4 as pluripotent markers in the ARXnCFP/nCFP iPSC at pluripotency stage.
Multi-lineage potency assay of ARXnCFP/nCFP iPSC line.
Differentiation Potency
Microbiology / Virus Screening |
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| Mycoplasma | Negative |
Genotyping
Karyotyping (Cell Line) |
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| Has the cell line karyotype been analysed? |
Yes
46, XX
Passage number: 25
Karyotyping method:
G-Banding
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Other Genotyping (Cell Line) |
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Genetic Modification
| Genetic modifications not related to a disease |
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