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UK Health Security Agency

HeLa mCherry-Histone H2B EGFP-Alpha Tubulin

HeLa mCherry-Histone H2B EGFP-Alpha Tubulin

Catalogue No.

17100525

Cell Line Name

HeLa mCherry-Histone H2B EGFP-Alpha Tubulin

Cell Line Description

The human histone H2B gene was fused to the gene encoding mCherry and Alpha Tubulin was similarly fused to EGFP. Both constructs were transfected into human HeLa cells to generate a stable line constitutively expressing H2B-mCherry and EGFP-Alpha Tubulin. The mCherry-Histone H2B fusion protein was incorporated into nucleosomes without affecting cell cycle progression. The cell line allows high-resolution imaging of both mitotic chromosomes and interphase chromatin. HeLa cell lines stably expressing GFP-tagged alpha-tubulin have been selected using 1.0 µg/ml puromycin and mCherry-tagged histone H2B selected using 2.0 µg/ml blasticidin.

General Info

Species

Human

Unique to ECACC

Yes

Also Known As

HeLa S3 EGFP-alpha-tubulin/H2B-mCherry

Release Conditions

Restricted - commercial organisations are required to complete the 'Cell Line Release Authorisation for Research Use in Commercial Organisations' release conditions form in the supporting documents section.

Characteristics

Tissue of Origin

Cervix

Morphology

Epithelial

DNA profile (STR Profile)

Amelogenin: X

CSF1PO: 9, 10

D3S1358: 15, 18

D5S818: 11, 12

D7S820: 8, 12

D8S1179: 12, 13

D13S317: 12, 14

D16S539: 9, 10

D18S51: 16

D21S11: 27, 28

FGA: 18, 21

Penta D: 8, 15

Penta E: 7, 17

TH01: 7

TPOX: 8, 13

vWA: 16, 18

Applications

To investigate the mechanisms of cell division, two HeLa cell lines stably expressing EGFP-tagged histone H2B and mCherry-tagged histone H2B -EGFP-tagged alpha-tubulin were generated in which the genetic material and the microtubule strings have been made fluorescent so that they can be observed while cells divide under the microscope.Specifically the cells were used to determine if PP6 catalytic activity is required for normal nuclear morphology, mitosis and cytokinesis. These cells were used to reveal that PPP6C-depletion in cells does not form a bipolar spindle with normal kinetics and results in failure to efficiently align chromosomes at the metaphase plate.

Disease

Cancer

Culture Conditions

Cell Type

Epithelial

Subculture Routine

Trypsinise cultures at ~70-80% confluence with 0.05% Trypsin/EDTA. Seed flasks at 1-3 x 10⁴ cells/cm². Cultures must be incubated in a humidified 10% CO₂/90% air incubator at 37°C. Note: The depositor had grown these cells at 5% CO₂. Here at ECACC we grow cells at 10% CO₂ when cells are grown in DMEM media.

Culture Medium

DMEM + 2mM Glutamine + 10% Foetal Bovine Serum (FBS).Recommend used of selection antibiotics (1µg/ml Puromycin and 2µg/ml Blastocidin) for maintenance of expression. The transgene is stably integrated into the genome of the cells so for short term culture it is not strictly necessary to include the antibiotics.

Growth Mode

Adherent

Additional Info

Depositor

Ximbio (formerly known as Cancer Research Technologies Limited)

Country of Origin

United Kingdom

GMO Status

Genetically Modified Organism Class 1 (GMO1)

Genetic Modification

The human histone H2B gene was fused to the gene encoding mCherry and Alpha Tubulin was fused to EGFP. Both were transfected into HeLa cells to generate a stable line expressing H2B-mCherry and Alpha Tubulin.Expression constructs were produced in pcDNA3 vectors modified to encode the chicken -actin promoter to reduce expression levels, the required tags (EGFP or mCherry) and the puromycin, or blasticidin resistance selective markers.

Hazard Group (ACDP)

Hazard Group (ACDP) 2

Applications

References

Nunes Bastos, R and Barr, F.A. Plk1 negatively regulates Cep55 recruitment to the midbody to ensure orderly abscission J Cell Biol. 2010 (Nov) 191(4): 751-760. PMID: 2983065 Dunsch, A.K et al. Dyenin light chain 1 and a spindle-associated adaptor promote dyenin asymmetry and spindle orientation. J Cell Biol. 2012. 198(6): 1039-1054. PMID: 22965910. Zeng., K. et al. Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2. J Cell Biol. 2019 (Mar).191(7): 1315-1332. PMID: 21187329.

Available Formats

  • Frozen

If use of this culture results in a scientific publication, it should be cited in the publication as: HeLa mCherry-Histone H2B EGFP-Alpha Tubulin (ECACC 17100525).

Unless specified otherwise, at ECACC we routinely handle all of our cell lines at containment level 2 in accordance with the ACDP guidelines (Advisory Committee on Dangerous Pathogens) (UK). All cell cultures have the potential to carry as yet unidentified adventitious agents. It is the responsibility of the end user to ensure that their facilities comply with biosafety regulations for their own country. ACDP Guidance: Biological agents: Managing the risks in laboratories and healthcare premises.

The Culture Collections represent deposits of cultures from world-wide sources. While every effort is made to ensure details distributed by Culture Collections are accurate, Culture Collections cannot be held responsible for any inaccuracies in the data supplied. References where quoted are mainly attributed to the establishment of the cell culture and not for any specific property of the cell line, therefore further references should be obtained regarding cell culture characteristics. Passage numbers where given act only as a guide and Culture Collections does not guarantee the passage number stated will be the passage number received by the customer.

Cultures supplied by Culture Collections are for research purposes only. Enquiries regarding the commercial use of a cell line are referred to the depositor of the cell line. Some cell lines have additional special release conditions such as the requirement for a material transfer agreement to be completed by the potential recipient prior to the supply of the cell line. Please view the Terms & Conditions of Supply for more information.