Haplobank methods collection

doi:10.1038/protex.2017.104

Method Article

Haplobank methods collection

https://doi.org/10.1038/protex.2017.104

This work is licensed under a CC BY 4.0 License

This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.

Version 1

posted

You are reading this latest protocol version

Large scale, genome wide datasets revolutionize our understanding of biological processes and disease mechanisms, but they also reveal a high degree of variance in functional and phenotypic studies due to genetic and epigenetic differences in cell populations. This has resulted in many studies that could not be repeated, a key issue for biomedical research. The ability to unambiguously annotate genes to phenotypes has therefore become of paramount importance. Utilizing multiple mutagenesis systems that can deliver large vectors to haploid murine embryonic stem cells (ESC), we now developed a resource of more than 100,000 murine ESC clones with targeted, barcoded mutations targeting almost 17,000 genes. This represents the largest homozyogous mutant ES library available to the scientific community to date. Importantly, these clones are conditional knockouts that allow for induction of mutations as well as reversal of mutants and thus side-by-side comparison of mutant and repaired target genes directly in sister cells. Our system not only avoids off-target effects, but also confounding background effects, and therefore improves reproducibility. Our new resource enables the comprehensive and robust interrogation of the genome. All characterized mutant ES cell clones and genome-wide saturating, repairable libraries we have constructed are shared with the entire research community.

This collection of cell culture- as well as molecular protocols aims to support the use of haploid mutant cell lines for rigorous genetic studies.

Cell biology

Developmental biology

Genetics

Biotechnology

Biological techniques

mouse ES cells

stem cells

haploid

gene trap

biobank

angiognesis

Gene trap mutagenesis in haploid mouse embryonic stem cells (ES cells) allows generation of hemi-/homozygous revertible mutations. Haplobank is a collection of over 100 000 mapped mutations shared with the research community.

This protocol collection of cell culture- and molecular techniques accompanying the publication of Haplobank in Nature (Elling, Wimmer et al., 2017, in press) aims to support use of cell lines for rigorous genetic analysis.

All available cell lines can be browesed and targeted and validated ES cells can be obtained from www.haplobank.at .

Oligonucleotide sequences for validation are supplied with the cell line.

Additional reagents required depend on the application, ES cells grow feeder free in standard serum-LIF conditions as well as in 2i. A complete protocol booklet is available as supplementary file.

Equipment needed for cell culture experiments include a standard cell culture setup (sterile hood, centrifuge, microscope, cell culture dishes, pipettes, media) as well as a standard wet lab setup including PCR machines and centrifuges.

To order Haplobank cell lines, please register at www.haplobank.at.

For all detailed protocols concerning use of the clone collection, please download the supplementary file "haplobank booklet".

We aim for a turnaround time of 1-1.5 months to ship clones. As they are already expanded, you will be able to initiate experiments directly after cells recovered from thawing, typically 2 days after receiving the shipment.

Should you experience unexpected results or have questions with the protocols, please contact the Haplobank team at office_at_haplobank.at.

Due to expected clonal variability (see Elling, Wimmer, et al., Nature 2017, in press) we recommend to study phenotypes in sense ans antisense orientation side by side. Furthermore, we suggest to also use the wt parental cell line as control.

Effect of gene trapping is not always predictable for every genomic locus, therefore we recommend qPCR with primers in exons adjacent to the gene trap vector or western blot to confirm loss of function.

Derivation of haploid embryonic stem cells: Elling et al. 2011, doi: 10.1016/j.stem.2011.10.012

Review on haploid genetics: elling et al., 2014: doi: 10.1016/j.febslet.2014.06.032

Haplobank clone collection: Elling et al., 2017, Nature in press

We thank all members of our laboratories for reagents, experimental support and critical discussion. We thank the IMBA/IMP service facilities, as well as the VBCF for technical support. Haplobank is generously funded by Nestlé Institute of Health Science NIHS as well as the Austrian National Bank (OeNB) and Era of Hope/National Coalition against Breast Cancer/DoD. U.E. is supported by the Austrian Academy of Sciences, the Austrian National Bank (OeNB), and is a Wittgenstein Prize fellow. D.B is supported by a FWF grant P23308-B13. J.M.P. is supported by an Advanced ERC grant and an Era of Hope/DoD grant. A.S. is supported by a European Research Council (ERC) Consolidator Grant (no. 647320) awarded to A.S., Boehringer Ingelheim GmbH, and the Austrian Research Promotion Agency (FFG).

The authors confirm, that there is no conflicting financial interests.

supplement0.docx
Protocol collection Haplobank protocols booklet This supplementary file contains all relevant information and protocols necessary for the use of the Haplobank collection.

Download PDF

Version 1

posted

You are reading this latest protocol version

Haplobank methods collection

Status:

Version 1

Abstract

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Haplobank methods collection

Status:

Version 1

Abstract

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Manage Cookie Preferences