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What is the Allen Cell Collection?
The Allen Cell Collection is a bank of human induced pluripotent stem cells 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 maintained and administered 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 GFP-tagged 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 on the Allen Institute for Cell Science Cell Collection page at http://www.allencell.org/cell-line-catalog.
Will these lines be available with other tags (different fluorescent protein colors, Flag, SNAP, Halo…)?
The inaugural collection is exclusively green fluorescent protein tagged (EGFP or mEGFP), but there are red fluorescent protein tagged lines in the development pipeline, which will become available in 2017. The focus of the Allen Institute for Cell Science’s research is live cell imaging so the Allen Cell Collection will only offer FP tagged lines, 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 their Cell Collection page at http://www.allencell.org/cell-line-catalog.
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 all of these studies are provided in the Certificate of Analysis for each line here on the Coriell site and are also available on the Allen Institute for Cell Science Cell Collection page at http://www.allencell.org/cell-line-catalog. 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 on the Allen Institute for Cell Science Cell Collection page at http://www.allencell.org/cell-line-catalog and the donor plasmids will be available from the nonprofit plasmid repository, Addgene, by Q2 of 2017.
Are steps taken to determine whether genomic stability of the iPSC line is effected by the editing/culturing process?
Yes, karyotype analysis is performed as a routine part of the QC 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 available on the Allen Institute for Cell Science Cell Collection page at http://www.allencell.org/cell-line-catalog.
Is off-target editing checked?
Yes, PCR amplification and Sanger sequencing of up to 10 predicted sites (using CasOFFfinder) is performed for each CRISPR per clone.
Is deep sequencing performed?
Yes, RNAseq and exome seq is performed on the final gene edited clones. This data will be shared by the Allen Institute for Cell Science once an efficient and effective method for sharing has been identified.
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. Extensive quality control data is available and in some cases additional functional studies contributed by collaborators. Visit their Cell Collection page at http://www.allencell.org/cell-line-catalog to learn more.
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 catalogue pages and it is also available on the Allen Institute for Cell Science website along with other useful information to help you get these cells flourishing in your own laboratories (http://www.allencell.org/cell-line-catalog).
How do you image the labelled structures in these endogenously tagged cells?
The GFP-tagged proteins are expressed endogenously and therefore may not appear as bright as they would in an overexpressed system. For imaging we plate cells onto matrigel-coated high-quality glass bottom coverslips (Cellvis) and image cells in phenol-free mTeSR media (StemCell Technologies). Our most common microscope configuration are a Zeiss spinning disk fluorescence microscope with a Yokogawa CSUX1 head, Hamamatsu CMOS camera, and a 488 laser (GFP). 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 below entitled, " 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 $600 per fluorescently tagged hiPSC vial for academic or non-profit entities.
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 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.