Real-Time Quantitative PCR-based System for Determining Transgene Copy Number in Transgenic Animals

Abstract

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.

Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

“…PCR-based methods, while limited in scope to analyze large-scale transgene structure, can be useful for estimating transgene copy number. Quantitative PCR (Q-PCR) can also be easily applied to many DNA samples in parallel and provides results faster than traditional Southern blotting, with similar accuracy as we and others have shown (Ballester et al 2004). …”

“…Unlike the method described here, some previous studies describe real-time PCR methods to estimate copy number on liver biopsy DNA samples, requiring the sacrifice of a transgenic mouse (Ballester et al 2004). Therefore, our method is advantageous for use with valuable transgenic mice (such as founders) for which premature sacrificing is undesirable.…”

“…In addition, our method estimates copy numbers based on a standard curve of known BAC copy number standards. Many other studies have used the 2 −ΔΔCt method to calculate copy number (Ballester et al 2004;Tesson et al 2002) which requires nearly equivalent PCR efficiencies between the unknown samples and the control. Others have reported that phenol/chloroform-extracted tail DNA samples contain PCR inhibitors (Burkhart et al 2002) or cannot be quantified accurately by UV spectrophotometry due to phenol contamination (Alexander et al 2004).…”

Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo

“…DNA was isolated from tail clips. Relative quantification of gene target VMAT2 and a reference gene, Tfrc, was obtained using the GoTaq qPCR Master Mix (Promega) on an Applied Biosystems 7500 Real-Time PCR System (55). Allelic copy number of VMAT2 was determined by the comparative C T method.…”

How much is too much? Phenotypic consequences of Rai1 overexpression in mice

“…A single-copy gene, glucagon, was used as an endogenous control with the primer/probe set sequences described previously. 16 All samples were run in triplicate in 20-ml reactions containing 2 Â TaqMan Universal PCR Master Mix (Applied Biosystems), primers at a final concentration of 900 nM each, 250 nM of the probe, and 50 ng of DNA. The PCR was run in the ABI Prism s 7900 HT Sequence Detection System (Applied Biosystems) using the following amplification parameters: 10 min at 951C, and 40 cycles of 15 s at 951C and 1 min at 601C.…”