FAQ

What is the Allen Cell Collection?

The Allen Cell Collection is a bank of human induced pluripotent stem cells (hiPSCs) that are currently derived from the WTC parental line released by the Conklin Laboratory at the J. David Gladstone Institute (available through Coriell as GM25256, where you can also find the de-identified patient information associated with the line). The WTC parental line (GM25256) was derived episomally from a healthy donor and has been fully sequenced, has a normal karyotype, differentiates into multiple cell types, and is amenable to gene editing. Using CRISPR/Cas9, these cells have been endogenously tagged with a fluorescent protein allowing researchers to see individual structures inside of healthy, living human cells with astonishing clarity. The Allen Cell Collection is distributed by the Coriell Institute for Medical Research (Coriell).

Will the Allen Cell Collection expand to use other iPSC lines?

Yes, but not at this time.

Why are these particular proteins being tagged?

The goal of the Allen Institute for Cell Science is to understand cellular organization and dynamics using live cell imaging. To achieve this, proteins, that represent key cellular structures commonly studied by cell biologists, are being tagged with fluorescent proteins to serve as reporters for the locations of these structures during imaging studies.

What determines what proteins get tagged and therefore what lines are available in the Allen Cell Collection?

In order to be considered for tagging a protein must represent a key cellular structure of interest to the cell biology community. The protein that best represents that structure and works well for imaging is identified from the literature and through engagement with members of the cell biology community who have direct experience with the structure of interest. The current list of structures for which reporter lines are being generated is listed in the Cell Catalog on the Allen Cell Explorer website, allencell.org.

Will these lines be available with other tags (different fluorescent protein colors, Flag, SNAP, HaloTag, etc.)?

Most of our single edited lines are mEGFP-tagged but there have a few single edited lines which are mTagRFP-T-tagged. We have a few dual lines with both a mEGFP and mTagRFP-T and a triple line which has an additional HaloTag. The focus of the Allen Institute for Cell Science’s research is live cell imaging so the Allen Cell Collection will only offer lines compatible with live imaging, however, the protocols that are available on their website can be used to introduce any tag into the genome. For a full list of the structures currently being tagged by the Allen Institute for Cell Science visit the Cell Catalog on the Allen Cell Explorer website, allencell.org.

What steps are taken to verify that the tag does not adversely affect the function of the protein?

Several quality control assays are performed to test for obvious adverse effects, including the localization of the protein to the correct structure, cell and organelle morphology, growth rate, and differentiation. The results of these control assays are provided in the Certificate of Analysis for each line here on the Coriell site and are also available in the Cell Catalog on the Allen Cell Explorer website, allencell.org. However, in depth functional assays specific to each structure are not performed as this is currently beyond the scope and expertise of the Allen Institute for Cell Science project. The research community is encouraged to do these studies and share their results.

Are the CRISPR sequences and plasmids that resulted in successful editing available?

Yes. The CRIPSR sequences that resulted in successful editing are available in the Cell Catalog on the Allen Cell Explorer website, allencell.org and the donor plasmids are available for academic and non-profit institutes from the non-profit plasmid repository, Addgene.

Are steps taken to determine whether genomic stability of the iPSC line is affected by the editing/culturing process?

Yes, karyotype analysis is performed as a routine part of the quality control process to address the genomic stability of the lines. These results are available in the Certificate of Analysis for each line here on the Coriell site and are also in the Cell Catalog on the Allen Cell Explorer website, allencell.org .

Is off-target editing checked?

Yes, PCR amplification and Sanger sequencing of up to 10 predicted sites (using CasOFFfinder) was performed for the first 11 lines. Since no off-target editing could be detected, this step is no longer a part of the quality control workflow.

Is deep sequencing performed?

Yes, RNA-seq and exome sequencing is performed on the released lines. The exome and transcriptional profiles of the different lines can be found on the Genomics page on the Allen Cell Explorer website, allencell.org.

Is there more data available on these cell lines?

Yes, the Allen Institute for Cell Science is using these lines in their research and they have posted a growing body of data on their Allen Cell Explorer website, allencell.org. In addition, extensive quality control data and in some cases additional functional studies contributed by collaborators can be found in the Cell Catalog on the Allen Cell Explorer website.

What assistance is available for working with these hiPSC lines?

The Allen Institute for Cell Science’s Culture Protocol is provided as a link on all the cell line catalog pages on Coriell. In addition detailed protocol and video tutorials about thawing, maintaining, banking and plating the cells for imaging can be found on the Method & SOPs page on the Allen Cell Explorer website, allencell.org. Cell culture related questions can be also posted in the Allen Cell Discussion Forum on allencell.org.

How do you image the labelled structures in these endogenously tagged cells?

The labeled proteins are expressed endogenously and therefore may not appear as bright as they would in an overexpressed system. For imaging cells are plated onto phenol-free matrigel-coated high-quality glass bottom plates (Cellvis) and imaged in phenol-free mTeSR media (StemCell Technologies). The Institute’s most common microscope configuration is a Zeiss spinning disk fluorescence microscope with a Yokogawa CSUX1 head andHamamatsu CMOS camera. Cells are imaged either with a 20x 0.8NA objective for lower magnification or 100x 1.25NA water immersion objective for higher magnification, at 37°C and 5% CO2 in a temperature-controlled chamber. The approximate laser power measured at the sample for our standard 100x images is ~2.5 mW.

What do I need to do in order to access a line from the Allen Cell Collection?

Every requestor must execute a simple material transfer agreement and complete the online ordering process.

MTA is Downloadable here:

Commercial entities and academics or non-profits engaged in commercial activities should refer to the section, "How can I get access to these lines if I work at a commercial company or I am engaged in commercial activities at an academic or non-profit institute?"

What are the fees for access to lines in the Allen Cell Collection?

There is a fee of $650 per vial for academic or non-profit entities.

Can I share the lines once I have them up and running in my laboratory?

No, the terms of the UBMTA do not give an individual investigator the right to further distribute the lines. However, core facilities can become a Cell Science Approved Core Facility which can subsequently distribute the lines to investigators within same institution. Please refer to the next two sections on how to become and distributes lines as a Cell Science Approved Core Facility

Can my Institute’s core facility purchase the lines and distribute them to me and other investigators at our Institution?

Yes, if the core facility is an Allen Institute for Cell Science Approved Core Facility and the lines are purchase into the facility under the Core Facility MTA then the core can scale up the lines for banking and subsequent distribution to investigators within your institution. However, certain quality control criteria must be met by the core facility before distribution can occur and they must be shared with investigators using an MTA at least as restrictive and the UBMTA for individual investigator use, that is available through Coriell.

How does my Institution’s Core Facility become an Allen Institute for Cell Science Approved Core Facility?

To be consider as an Allen Institute for Cell Science Approved Core Facility please contact distributionreports@alleninstitute.org providing the following information; Name and contact information of Core Facility Director. A member of the Allen Institute for Cell Science team will contact you to outline the process.

How can I get access to these lines if I work at a commercial company or I am engaged in commercial activities at an academic or non-profit institute?

If you work at a for-profit company or an academic or non-profit entity wishing to engage in commercial activities with these cell lines, please contact the Coriell Customer Service. Commercial activity by an academic or non-profit entity is defined as fee-for-service or sponsored research or other arrangements in which another party has rights to the output of the work performed, even if the work is done by a non-profit. The Allen Institute for Cell Science is open to making their biological materials available to commercial entities for internal research purposes, but please be advised that third-party restrictions on component materials may impact their ability to share certain biological materials.