Greg Sykes, Robyn Hedges, Ming Hui, and Yvonne Reid STR Analysis: From Cells To Data Cultured Cells Import Into In-House Database for Comparison (Paradox macro) DNA Extraction (MoBio UltraClean DNA Bloodspin Kit, FTA Paper) GeneScan 3.1 and
Genotyper 2.0 Analysis (peak detection and labeling) PowerPlex 1.2 PCR (Promega) Electrophoretic Fractionation and Fragment Detection (ABI 310) Table 1. The Promega PowerPlex 1.2 STR Loci STR Locus Amelogenin
CSF1PO D5S818 D7S820 D13S317 D16S539 TH01 TPOX vWA Chromosomal Location Xp22.10-22.3 and Y 5q33.3-34 5q21-q31 7q
AATG AGAT Probability of a random match is lower than 1 in 108. ATCC holds over 2,000 human cell lines. Cell Line Workflow and How It Relates To the STR Analysis Depositor Material/ Token Freeze Master Cell Bank (Seed Freeze)
Working Cell Bank (Distribution Freeze) Profile Baseline Designated as oldest, most original material Cross-compared against all existing profiles at ATCC Comparison Profiles Cross-compared against all profiles of that cell line Unlike STR profiles generated from normal
human material, valid data from tumor cell lines are more complex due to: Heterogeneous original population (differences between cancer cells within the same mass) and subsequent selection. Addition or loss of chromosomal material due to uneven karyokinesis and cell hybrids. Mutation events occurring within the DNA sequence. Genetic drift with subsequent culture expansions. Figure 1. Example of a standard karyotype from CCL-75 (WI 38), normal lung tissue (n=46) Figure 2. Example of a karyotype from CRL-2061 (SJRH30), a
rhabdomyosarcoma cell line (n=84) Figure 3. This relatively balanced-peak electropharogram of a colon adenocarcinoma cell line is atypical of cell line profiles. Figure 4. Most tumor and transformed cell line profiles have unbalanced peaks, but the data are reproducible. This example happens to be from a liver adenocarcinoma. Figure 5. This sample was submitted to the ATCC as a cancer line coming from a single patient. With six loci having trisomies or more, this line is considered cross contaminated. The amelogenin locus is useful in cell
culture profiles: Finding appropriate X or X, Y peaks confirms the gender of the cell line (when provided). Female-derived cell lines with X, Y profiles are investigated; if the Y cannot be explained, the line is failed (not accessioned). Results From Chromosome Rearrangement Y Chromosome Figure 6. Based upon classic cytogenetic testing, cell line HTB-144 (JAR) was labeled female. STR analysis detected Y amelogenin. FISH analysis confirmed translocated Y chromosome material.
Cell lines submitted as male but lack Y amelogenin are documented and approved because: A deletion on the Y chromosome amelogenin primer binding site may prevent a strong (or any) signal. Certain cancers (e.g., bladder, renal, prostate, stomach, some leukemias) are more prone to a loss of the Y chromosome. Y chromosome is absent in up to 85% of the bone marrow cells of normal elderly males. Parental
Figure 7. Profile differences may emerge between normal tissue and tumors from the same patient or between a parental and derived cell lines. These differences may include: Nothing Loss of heterozygosity (including locus drop-outs) Gain of heterozygosity (somatic mutations) Parental: scrape Derived: trypsin
Figure 7. Profile differences may emerge between normal tissue and tumors from the same patient or between a parental and derived cell lines. These differences may include: Nothing Loss of heterozygosity (including locus drop-outs) Gain of heterozygosity (somatic mutations) Parental Derived
Figure 8. Compared to the parental cell line CRL-2570 (A3), a microvariant emerges in a derived line, CRL-2571 (I 9.2), following exposure to the frame-shifting mutagen, ICR-191. Unstable Alleles A B C D P13
E P13 F P11 G P13 H
MASTER P8 I P15 J P13 P13
K P13 P14 L P11 P14 Figure 9. Cell lines rarely exhibit allelic instability. This
umbilical cord line, CRL-1730 (HUV-EC-C), is an exception. Allele 9 at D13S317 has different intensities throughout the individual lots. However, the intensity of allele 9 within each lot is reproducible (e.g., lot A = weak 9; lot B = strong 9; lot D = absent 9). Pn is the passage number. Post-Electropherogram Interpretation and Data Analysis Computer analysis cross-compares new baseline data with all previously generated profiles. Working stock profiles are imported and checked against all earlier profiles of that cell line. Cell lines with at least two independent analyses are posted on the ATCC website (http://www.atcc.org/Products/str.cfm).
Commercially available kits enable research institutions, collections, patent offices, and scientists to confidently confirm or dispute cell line purity and authenticity. Summary Prior to their accession and distribution, ATCC uses STR analysis to screen all human cell lines for authenticity and purity. Tumor cell lines are unlike healthy tissue living within an organism. Most cell lines have undergone genetic mutation events. Abnormal karyology and sequence mutations impact the STR profile. Tumor and transformed cell line data interpretation is more challenging than that from normal material, but
the data are reproducible. Resulting STR data can be globally communicated to researchers. Acknowledgements Robyn Hedges Ming Hui Yvonne Reid [email protected] Ed Cedrone Scott Durkin Brett Hankins Qassim Azizi Kristen Mundy Ruth Monk The most exciting phrase to hear in science,
the one that heralds new discoveries, is not "Eureka!" (I found it!) but "That's funny." Isaac Asimov
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