Description: HeLa cells transduced with Retrovirus to stably express H2B tagged with GFP, from pWZL backbone, and sorted for low expression, the cells contain E6 protein which degrades p53, functionally knocking it out.

Description Key Words: HeLa H2B GFP tagged

Also Known As: HeLa^H2B-GFP Hela_H2B-GFP

Organism: Human (Homo sapiens) 

Strain: N/A

Tissue: Cervix cervical carcinoma

Growth Properties: Adherent

Morphology: Epithelial

Growth Medium:

DMEM – high glucose, 10% BGS, 1% NEAA, 1% Glutamax

100ug/mL Hygromycin.

Resuscitation: Remove protective cryoflex layer around the ampoule prior to thawing. Thaw the ampoule by gently agitating in a 37°C waterbath; thawing should be rapid (around 2 minutes). A centrifugation step to remove the cryoprotectant after thawing is necessary for this cell line. Recovery from thaw may take 2 days.

Subculturing Procedure:

Medium Renewal: 2-3 times per week. 

Subcultivation Ratio: 1:8 – 1:16 Seeding density 0.8 – 1.0 x10⁴cells/cm². Split subconfluent cultures (70-80%). Harvest the cells using 0.05% Trypsin/EDTA at 37°C for 5 minutes.

Culture Conditions: Incubate the culture at 37°C with 10% CO₂.

Cryoprotectant Medium: 10% DMSO + 90% FCS

Handling Procedure for Frozen Cells: Upon receipt, frozen ampoules should be transferred directly to liquid nitrogen storage without delay, if not to be used immediately. Storage at -80°C may result in loss of viability.

Additional Information: One of a pair of HeLa derivatives genetically modified to carry fluorescently tagged histone H2B proteins. H2B is a core component of the nucleosome, where it plays an important role in transcriptional regulation, DNA repair, and other essential cellular functions. This cell line, HeLa H2B-GFP, carries H2B tagged with eGFP. The second cell line, HeLa-2FP, carries both a H2B tagged with eGFP and H2B tagged with mCherry. Expression of both proteins enabled study of chromatin compaction in live cells using FLIM-FRET microscopy. The cell lines were published in PNAS in 2019, where they were used to study the remodelling of chromatin architecture at double-strand break sites during DNA repair.

Depositor: Tony Cesare, Children's Medical Research Institute, Australia

Reference: Lou J et al. Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response. Proc Natl Acad Sci U S A (2019) 116(15): 7323-7332. doi: 10.1073/pnas.1814965116.

PMID: 30918123

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