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

Abstract

Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used Taqman real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method, and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.

Real-time in vivo imaging of p16Ink4areveals cross talk with p53

“…To examine the copy number of the transgene, genomic DNA was prepared from early passage mouse embryonic fibroblasts (MEFs) derived from the p16-luc mice. The DNA sequence specific to this human chromosome segment was amplified and quantified by real-time PCR analysis (Ballester et al, 2004;Chandler et al, 2007) using genomic DNA prepared from the same number of early passage human diploid fibroblasts (HDFs) as a control. The signals obtained from the p16-luc MEFs were almost half compared with those from HDFs, which have two copies of the human Ink4a/Arf gene (Fig.…”

Copy number and integration sites in growth hormone transgenic goats

“…High copy number tandem integration is thought to lead to transgene silencing (Tang et al, 2007), and high copy numbers may decrease in aging transgenic animals. A previous study showed that at least approximately 10 copies were ideal for analysis (Chandler et al, 2007). In our study, 12 copies of the GH transgene ensure GH expression in transgenic goats.…”

“…We generated several transgenic goat lines by SCNT using the pcGH construct. Because the GH transgenic line was not generated by site-specific recombination technology, the integration sites and copy numbers were uncertain, and these factors may influence the phenotype of transgenic animals (Chandler et al, 2007). Recently, because less DNA is required and because of its high sensitivity, precision, and repeatability, quantitative PCR has replaced Southern blotting for determining copy number in transgenic animals (Ballester et al, 2004;Lipinski et al, 2012).…”

Selective Manipulation of Neural Circuits

“…The positional transgenesis approach, knock-in, inserts transgene into mouse genome at a specific location, such as Rosa26 and HPRT locus, while random transgenesis, which usually uses bacterial artificial chromosome (BAC), integrates the transgene at a random location. These days, the BAC transgenic approach has been widely used owing to its advantages, including ease of use and the availability of large library [32][33][34][35][36][37]. However, endogenous gene expression can be altered because the delivery of BAC may contain coding regions or promoters of other genes [20,38].…”