The Stars of Single Tandem Repeat
In identifying human and animal cell lines, it’s all about the traceability
The genetic analyzer machines churn out DNA sequencing data as sweet as supervisor Jeff Rabenstine’s singing. But it is not the ONLY tried and true Sanger Sequencing methods keeping the instruments busy. There are other sets of experiments churning out useful data, too. Using the capillary electrophoresis instruments, and a computer Oracle database for analysis, Short Tandem Repeat or STR profiling experiments are being run by technician and colleague Haley Neff and crew to demonstrate sub-species level identification of cell lines. This is a DNA fingerprinting method.
Like the unique fingerprints that Haley leaves on the softball as she fires pitch after pitch, this PCR-based “DNA Fingerprinting” method uses target-specific fluorescently-tagged oligonucleotide primers which hone in on particular DNA regions where a variable numbers of STR repeats are found. After separation by capillary electrophoresis on a genetic analyzer instrument, one cell line may be shown to have 4 repeats, another may have 6, 9, 10, or whatever is found at that particular loci.
The genetic analyzer laser reads the fluorescent molecular tags that get incorporated into the PCR-amplified pieces of DNA and records a colorimetric value and quantity in the readout, based on the size of the amplified material. Not just one region is studied, but multiple regions are tested at the same time—this is called a multiplex PCR. The level of discrimination increases by investigating more regions. We at CRL use both commercial “over the counter” kits (human cell line analysis) and custom-made materials. One multiplex PCR reaction may look for targets across the whole genome—8 or 16 or 21 different targets—or just a few, and the instrumentation compiles data from it all, giving a unique fingerprint to the cell line.
Speaking of fingerprints… If you remember where you were standing when the OJ Simpson verdict was read (I was holding a scoopula by the analytical balances in graduate school), then you know how tricky it was (and still is) to obtain solid evidence that holds up in court – or anywhere for that matter. It is all about the traceability and confidence in the data. As Johnny Cochran reminded the jury in the highly televised trial: “If it doesn’t fit… You must acquit!”
DNA studies are used as solid evidence in forensics and paternity testing. Our biologics manufacturing industry uses the same principles for cell line analysis. Show the data!
Cell lines are used by our clients to produce important biologics. Where they obtained those materials and how they were handled turns out to be really important. Traceability comes into play, again and again, not only in handling the material and labelling it properly, but because cell lines are live biological evolving organisms. Their genomes can be modified in day to day passages through cell culture. Even contamination splashes of HeLa cells have happened in historical laboratory snafus.
The famous HeLa cell line, credited for major contributions in cancer studies, has been cultured in laboratories ever since being derived from the early 1950’s cervical cancer biopsy of Henrietta Lacks, whose family has been in a many year legal battle over the credit given as well as requesting financial payback from the use of her cells. Because of the Human Papillomavirus 18 infection, these cells have a tremendous ability to grow and became the first immortalized human cell line, which indicates their aggressive nature in growth. Instances of cross contamination, due to mishandling of cell cultures, have been documented, impacting scientific discoveries. Therefore any and all verifications of cell origins, cell types, and cell line stability are essential in the industry today.
Retracing a cell line’s footsteps
In our molecular-based laboratories, we determine the unique STR fingerprints of human and certain animal cell lines. Then for our client’s information, we will search the STR result against a database (internal or external) to look for a close match. For example, if the client is using a particular cell line, we can compare it to published references. Databases are only as good as the data entered, but thankfully they are getting better and better. Our scientists collaboratively have discussed database entries with the curators at NCBI, ATCC, etc to hone in on database suitability. A new tool, the CLASTR STR Similarity tool, supported by the Swiss Institute of Bioinformatics (SIB) Cellosaurus, aims to pull together STR profiles from all databases for scientists to query.
What is next? Not coming quite as fast as Haley’s softball pitches, but on the horizon for the Biologics Methods Development team at Charles River, is a new method for mouse cell line STR analysis and the development of a new cell line database curated by the National Institute of Standards and Technology (NIST) and National Center for Biotechnology Information (NCBI). In 2017 and 2018, the Methods Development team tested a mouse STR analysis method developed by NIST’s Jamie Almeida in collaboration with scientists at laboratories around the world.
The goal was to look for consistency, to determine consensus allele-calling in nearly 50 mouse cell lines and investigate causes for inter-laboratory variability. The cell lines with 98% allele agreement between laboratories are now compiled in a NIST/NCBI Mouse database. The work resulted in a 2019 publication.
And now, to take the analysis methods to an even higher level of consistency, the Methods Development group will test an “allelic ladder” in hopes that the biologics industry will gain information about interlab variability, eventually with hopes to see commercial production of a kit. As an allelic ladder establishes a profile for every possible STR count at each genome location tested, this allows for software optimization of reading the allele counts of unknowns. This is the same procedure that has allowed human forensics, paternity testing and cell line analysis to be more reproducible. Eventually we will have a foot up on offering these new tests to our clients.
Such small laboratory leaps, but impactful with the global effort to improve cell line identification to help sub-speciate and verify client material.