Genetic Engineering & Biotechnology News

NOV15 2017

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22 | NOVEMBER 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News scientific journals have revised their submis- sion guidelines for research grants and peer- reviewed articles to include requirements for authenticating cell lines. The effectiveness of these new policies may depend on how these organizations decide to enforce them, but they represent a much-needed step toward solving a problem that has plagued the scien- tific community for over 50 years. Short-Tandem Repeat Analysis: A Scientist's Insurance Policy Well-established technologies for authenti- cating human cell lines exist, but barriers to their use—including awareness, complacency, time, and cost—have frustrated efforts to get past the current cellular identity crisis. Cell line authentication advocate and global stra- tegic marketing manager at Promega, Gabri- ela Saldanha, Ph.D., offers her perspective to researchers who might list cost as a reason to skip an identity check: "[Cell line authentica- tion] is like an insurance policy. Nobody likes paying for insurance, but you never know when you're going to need it." Unfortunately, many biomedical researchers do not realize or acknowledge that they're working in a flood zone—where estimates for the total percent- age of cell lines that have been contaminated or misidentified range from 18% to 36%. The current gold standard for authenti- cating human cell lines, short-tandem repeat (STR) analysis, is an identification technique used by forensics labs during criminal in- vestigations. Only 2–5 base pairs in length, STRs are short, repeated sequences in DNA that provide a powerful tool for human iden- tification, because the number of repeated units can vary significantly between individ- uals. The location on a chromosome where an STR occurs is known as an STR locus, and each locus represents a different contour in an individual's genetic fingerprint. Do-it-yourself types can purchase STR assay kits designed for cell line authentica- tion for less than $14 per test from compa- nies such as Promega and Thermo Fisher Scientific. These companies have optimized fluorescently labeled primers to allow co- amplification of multiple STR loci in a single PCR reaction (Figure 1). For example, Promega's GenePrint ® 10 and GenePrint ® 24 Systems co-amplify am- elogenin, a marker that indicates an individ- ual's sex, plus 9 and 23 autosomal STR loci, respectively. After amplification, researchers can perform size- and color-based separation and detection of STR loci using capillary electrophoresis. Finally, they can generate an STR profile and compare it to a reference to verify the identity of their cell lines. Some researchers may lack experience with the molecular biology techniques needed for STR, or they may be unable to access the right equipment. These researchers may resort to sending samples to a service provider, such as Genetica (Figure 2), ATCC, or Multiplexion. Although scientists have cited cost, time, and delays in research as the top three barriers to cell line authentication, 4 these companies have worked hard to establish fast, affordable services. "[Genetica] offers several different STR re- port options, from basic Excel spreadsheets to formal comparative reports, to fit the budget of any research lab," said Erin Hall, director of cell line authentication services at Genetica, and customers can expect to receive their re- sults in as few as two business days. Genetica's standard cell line authentication service uses amelogenin to distinguish between cell lines derived from male and female individuals plus 15 STR loci to validate cell lines (Figure 2), and they recently added a marker to detect mouse DNA contamination. "The test can easily pick up a low level of mouse [DNA] contamination in a human specimen," said Hall, who explained that "this additional marker is especially helpful for researchers using mouse xenograft models of human tumors or those that are using both human and mouse cell lines simultaneously." Of Mice and Men: Authenticating Non-Human Cell Lines A significant number of scientific studies use non-human cells, which are just as suscep- tible to cross-contamination, misidentification, and mislabeling as those from human samples. Although Genetica's mouse marker can help identify cross-contamination between mouse and human cell cultures, a major limitation of STR analysis is that it's currently only avail- able for human cell line authentication. In an effort to build the infrastructure need- ed to enable the identification of non-human cell lines using STR analysis, a number of or- ganizations, including Genetica and ATCC, have joined the National Institute of Stan- dards and Technology to form the Mouse Cell Line Authentication Consortium (MCLAC). "Mouse is the next most popular cell line af- ter humans, so it's the logical extension of Raising the Bar for Cell Line Authentication Bioprocessing Feature Continued from page 1 Figure 2. An electropherogram of PowerPlex®16HS results, provided by Genetica, showing a mixture of cell line MDA-MB-231 (breast adenocarcinoma) and cell line A549 (lung carcinoma). The presence of more than two peaks at more than one genetic marker is a good indicator that a mixture may be present; the other potential cause of additional peaks is genetic instability, an occurrence that can be normal for certain cancer cell lines. In this example, note the presence of the "Y" chromosome (amelogenin marker; bottom panel); A549 is derived from a male individual, whereas MDA-MB-231 is derived from a female. This helps lead to the conclusion that a mixture is present, as opposed to genetic instability. Figure 1. Promega's GenePrint 10 System and GenePrint 24 System are optimized for amplification of purified DNA or direct amplification of DNA from storage card punches using the GeneAmp PCR System 9700 thermal cycler. Either approach may be used to identify STR loci, which consist of repetitive DNA sequences with varying numbers of repeats. Each STR locus can be amplified by PCR, and the amplified products can be labeled with different fluorophores, making the products easy to distinguish by size and color.

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