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  • Unfortunately existing cytogenetic karyotyping methods are l

    2018-11-12

    Unfortunately, existing cytogenetic karyotyping methods are labor intensive, requiring at least 15 fixed metaphase preparations, subsequent DAPI staining, and analysis with an epi-fluorescence microscope equipped with specialized digital imaging software. Conventional karyotyping is expensive (∼$400/clone) and often needs to be outsourced to specialized core facilities. Therefore, we designed a simple, fast, and cost-effective (∼$1/clone) SYBR-Green-I-based qPCR procedure to identify common chromosomal aneuploidies found in genetically manipulated mESCs. In SGI-1776 Supplier to regular PCR, qPCR combines PCR amplification and detection into a single step using detection chemistries such as target-specific hydrolysis probes and SYBR Green I dye, which binds double-stranded DNA and emits fluorescence only when bound. To date, qPCR is the benchmark for gene expression analysis, but recent developments also encourage the use of qPCR for copy number determination (D’haene et al., 2010b). In this regard, qPCR is currently used for diagnostic copy number profiling of the SHOX gene region in idiopathic short stature (ISS) and allied disorders (D’haene et al., 2010a). In addition, it has been shown to be an elegant tool to accurately screen for correctly targeted mESC clones (using the so-called loss-of-allele assay or LOA) (Frendewey et al., 2010) and for genotyping of gene-targeted and transgenic mice (Haurogné et al., 2007; Sakurai et al., 2008). These studies show that qPCR can be applied to discriminate 2-fold differences in the copy number of a specific transgene, allowing discrimination between homozygous and hemizygous transgenic animals. We have applied and optimized this method to discriminate among chromosomal aneuploidies, ranging from loss of a chromosome to extra copies of a particular chromosome of interest. The accumulation of fluorescent signal during the exponential phase of a PCR results in a fast, precise quantification of PCR products and allows for objective data analysis. The quantification cycle or Cq value represents the fractional PCR cycle that is characteristic for the amplification curve (e.g., where increase in fluorescence is maximum) or at which the fluorescence SGI-1776 Supplier crosses a certain threshold (D’haene et al., 2010b). Cq value and initial amount of input DNA are inversely related: a sample that contains more copies of template will cross the threshold at an earlier cycle compared to one containing fewer copies of template (Schmittgen et al., 2000). Consequently, the theoretical difference in Cq (dCq) between two and three copies of an autosome is about a half cycle, whereas the dCq between one and two copies is one cycle. With 53 mESC lines (all male), we were able to validate the sensitivity and accuracy of our approach in great detail, which resulted in a quick and user-friendly paradigm to test the quality of any genetically manipulated mESC clone in less than 1 day. We confirmed our approach with the two most common chromosome aneuploidies found in mESCs: chr8 and chrY, and we further validated the approach with rare chr11 and partial chr1 aneuploidies among our collection of cytogenetically karyotyped clones.
    Results
    Discussion We are able to rapidly identify the common Trisomy 8 and chrY aneuploidies as well as the rare Trisomy 11 and partial Trisomy 1 aneuploidies in mESCs. Conventional cytogenetic karyotyping requires direct inspection of chromosomal images through human visual interpretation. The SYBR Green qPCR-based assay is numerical with fixed confidence levels and not dependent on such subjectivity (Frendewey et al., 2010). Our method provides a standardized cutoff IC SD of 0.0906 by which all mESC lines can be determined as “normal.” This number might vary slightly, but will not exceed 0.1. A cumulative analysis of cytogenetic karyotyping from four groups, including ours, reveals that out of 355 mESC clones only 2 (0.5%) contained Trisomy 11 without accompanying aneuploidies (Liu et al., 1997; Sugawara et al., 2006). In our study, Trisomy 11 only occurred in the presence of Trisomy 8 (ESC 13, 16, 18). Therefore, screening for Trisomy 11 aneuploidy is redundant. In addition, these findings are consistent with our single ESC clone containing 31% Trisomy 1 cells without accompanying aneuploidies (ESC 8). In order to identify this 31% Trisomy 1 frequency in ESC 8, we had to cytogenetically karyotype 54 cells (Table S1). To confirm these data, we screened for partial Trisomy 1 with primers to olfr16 (located on chr1, Table 2) and show a NRQ value of 1.4, which represents an absolute chr1 copy number of 2.8 (data not shown), similar to where the NRQ value would lay for a 30% Trisomy 8 (Figure 3A). Our method is straightforward, and the Trisomy 1 data show the ease of adding extra primer sets to the analysis if necessary. In addition, the IC SD for ESC 8 including the chr1 analysis was 0.1699, which is greater than our cutoff of 0.0906, and clearly identifies aneuploidy. Importantly, adding the NRQ values for chr1 to the IC SD calculation for the calibrators (ESC 31, ESC 52, and ESC 53) maintained their IC SD values below 0.05 (data not shown).