hiPSC-ARX-T2A-H2B-CFP-Flag

General#

Cell Line

hPSCreg Name HMGUi001-A-4
Alternative name(s)
hiPSC-ARX-T2A-H2B-CFP-Flag
Cell line type Human induced pluripotent stem cell (hiPSC)
Last update 7th May 2020
User feedback
No feedback available yet.

Login to share your feedback, experiences or results with the research community.

Provider

Generator Institute of Diabetes and Regeneration Research (IDR)
Owner Institute of Diabetes and Regeneration Research (IDR)
Distributors
Derivation country Germany

External Databases

BioSamples SAMEA7002448

General Information

* Is the cell line readily obtainable for third parties?
No
Subclone of

Donor Information#

General Donor Information

Sex female
Ethnicity Caucasian

Phenotype and Disease related information (Donor)

Diseases No disease was diagnosed.
Disease associated phenotypes no phenotypes
Family history no
Is the medical history available upon request? no
Is clinical information available? no

Karyotyping (Donor)

Has the donor karyotype been analysed?
No

Other Genotyping (Donor)

Is there genome-wide genotyping or functional data available?
No

External Databases (Donor)

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

The source cell information can be found in the parental cell line HMGUi001-A.
Passage number reprogrammed 25

Reprogramming method

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

Other

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
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
Endoderm
Ont Id: UBERON_0000925
Other
Mesoderm
Ont Id: UBERON_0000926
Other
Ectoderm
Ont Id: UBERON_0000924
Other

Microbiology / Virus Screening

Mycoplasma Negative

Genotyping#

Karyotyping (Cell Line)

Has the cell line karyotype been analysed?
Yes
46, XX
Karyotyping method: G-Banding

Other Genotyping (Cell Line)

Genetic Modification#

Genetic modifications not related to a disease
ARX (target)
Transgene expression
Xp21.3
CRISPR-associated (CRISPR/Cas) System