title: Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.
abstract: Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.
authors.affiliation: Department of Surgery, East Carolina University, Greenville, NC, USA.
authors.forename: Molishree
authors.initials: M
authors.lastname: Joshi
authors.forename: H
authors.initials: H
authors.lastname: Keith Pittman
authors.forename: Carl
authors.initials: C
authors.lastname: Haisch
authors.forename: Kathryn
authors.initials: K
authors.lastname: Verbanac
mesh_terms: D019264:Adoptive Transfer / Q000379:methods; D000818:Animals; D018827:Carcinoma, Lewis Lung / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis; D042783:Endothelial Cells / Q000378:metabolism*; D018628:Gene Dosage; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D008113:Liver Neoplasms / Q000098:blood supply / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D004273:DNA, Neoplasm; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019914:Taq Polymerase