An improved method for determination of gene copy numbers in transgenic mice by serial dilution curves obtained by real-time quantitative PCR assay.
Precise characterization of transgene insertion is necessary for phenotype interpretation of transgenic animals. To check for the presence of deletions, estimate the number of inserted transgene copies, and in addition, identify the zygosity of transgenic mice, gene copy numbers were determined by real-time quantitative PCR. Instead of correlating tested samples to a single relative standard curve, serial dilution curves were constructed for every mouse sample. A novel statistical approach was designed in which mice with the same copy number were characterized by the adjusted group mean and standard deviation common to the target sequence. This enabled us to characterize the variability of the obtained results, statistically compare different groups of mice and estimate precision and limits of the applied method.
Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Salata 12, HR-10000 Zagreb, Croatia. dominic@mef.hr
Dinko
D
Mitrecić
Miljenko
M
Huzak
Marija
M
Curlin
Srećko
S
Gajović
D000704:Analysis of Variance; D000818:Animals; D003433:Crosses, Genetic; D004247:DNA / Q000378:metabolism; D017353:Gene Deletion; D018628:Gene Dosage; D005821:Genetic Techniques; D005838:Genotype; D006579:Heterozygote; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D015233:Models, Statistical; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019076:Transgenes
D004247:DNA