Chimerism Analysis in Sex-Mismatched Murine Transplantation Using Quantitative Real-Time PCR

Abstract

Murine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 102% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500600 reactions per day.

Myogenic Potential of Muscle Side and Main Population Cells after Intravenous Injection into Sub-lethally IrradiatedmdxMice

“…Real-time quantitative DNA amplification of Y-target sequences was performed using a modification of a previously described method (Byrne et al 2002). The mouse Y-chromosome specific primers sense (5 Ј -TGGAGAGCCACAAGCT-AACCA-3 Ј ) and antisense (5 Ј -TCCCAGCATGAGAAAGA-TTCTTC-3 Ј ) amplified an 81-nucleotide product specific for ZFY2 gene as described (Mardon and Page 1989;Byrne et al 2002).…”

“…For each sample, the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was also amplified as a loading control, using sense (5 Ј -GGAGATTGTTGCC-ATCAACG-3 Ј ) and antisense (5 Ј -GTCTCGCTCCTGGAA-GATGG-3 Ј ) primers, which generate a 140-nucleotide PCR product. ZFY2 primers were used at a final concentration of 300 nM for the sense and 900 nM for the antisense primer as previously described (Byrne et al 2002), whereas both GAPDH primers were used at a final concentration of 300 nM. Genomic DNA from test samples and standard control curve were amplified in the same reaction using the SYBR Green PCR reagent kit (Applied Biosystems; Foster City, CA) according to the manufacturer's protocol.…”

“…To obtain a more comprehensive assessment of the amount of male-derived cells that were present in the recipient mdx muscles 12 weeks after IV donor cell injection, genomic DNA extractions were performed on the remaining quadriceps and diaphragm samples that had already been subjected to FISH/immunohistochemistry analysis. To calculate the amount of donor-derived DNA present in these muscles, which would include both donor nuclei fused to myofibers and interstitial ones, real-time quantitative PCR was performed via amplification of a gene present on the mouse Y-chromosome, named ZFY2 (Mardon and Page 1989;Byrne et al 2002) and GAPDH internal control. Real-time quantitation was performed by monitoring the fluorescent emission of SYBR green dye bound to the PCR prod- ucts, using a modification of a previously described method (Byrne et al 2002).…”

Overview of Real-Time PCR Principles

“…Robust chimerism analyses with extremely large dynamic ranges based on insertion/deletion polymorphisms and on SNPs are also possible (Wilhelm, Reuter et al 2002;Maas, Schaap et al 2003). Genetic chimerisms have been monitored by Ychromosome-specific real-time PCR for sex-mismatched transplantations (Fehse, Chukhlovin et al 2001;Byrne, Huang et al 2002;Elmaagacli 2002) and by allele-specific realtime PCR (Maas, Schaap et al 2003;Shively, Chang et al 2003). This combination of allelespecific amplification with real-time PCR has been shown to reveal detection limits of down to 0.01% for SNPs (Maas, Schaap et al 2003).…”

Retrovirus molecular conjugates: a versatile and efficient gene transfer vector system for primitive human hematopoietic progenitor cells

“…Real-time PCR was conducted on a ABI PRISM 7700 Sequence Detector (PE Biosystems) as previously published in detail by our group. 14,15,24 The samples were analyzed with the SDS 1.9.1 software (ABI PRISM s sequence detection system 1.9.1, PE Biosystems, Foster City, CA) and corrected for total DNA content using a spectrophotometer (DU640B, Beckman Instruments).…”