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 that 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.

FIG. 1

BAC DNA copy number standards generate reproducible curves in real-time PCR. (a) Amplification plot depicting the Neo (blue) and Jun (purple) results for the copy number standards. As expected, each standard is approximately one cycle apart for the Neo assay and amplification plots are similar for all standards for the Jun assay. (b) Copy number standards were used to generate standard curves in real-time PCR using Neo and Jun primer/probe sets on two independent days (filled boxes = Day 1, empty boxes = Day 2). Replicate experiments indicate the copy number standards provide highly reproducible standard curves (R² = 0.9999, R² = 0.9976) for estimating copy number of experimental samples.

FIG. 2

DNA concentration has little impact on copy number estimates over a wide range of input DNA. DNA samples from two transgenic mice with copy number estimates of 2 (filled box) and 1(filled circle) were each used to create a 2-fold dilution series of DNA templates, such that 1–64 ng DNA (total input) from each mouse were subjected to real-time PCR. The amount of template DNA versus copy number estimations indicate copy number estimations vary little as input DNA ranged from 4–32 ng.

FIG. 3

Copy number estimates are consistent within independent transgenic lines. Shown are copy number estimates from individual mice, as determined from yolk or tail DNA samples of multiple progeny from eight independent Bmp4 BAC transgenic lines.

FIG. 4

Copy number estimation by dot blot hybridization. (a) Dot blot hybridized with a BAC transgene-specific probe (IRES-βgeo). (b) The same dot blot stripped and reprobed with a genomic control probe (mouse Gdf6 3’UTR fragment) to account for differences in input DNA. Standard curve (0–128 copies per diploid genome) and genomic DNA samples were assayed in triplicate. Copy number estimates for genomic DNA samples were derived by comparing the ratio of dot intensities for transgene-specific and control probe hybridizations in standard curve samples spiked with known quantities of pIBGFTet plasmid (see methods).

FIG. 5

The distribution of variation in copy number across stably breeding lines and transiently generated founder embryos or liveborn founder mice. While the majority of integration events contain 1–25 copies, all animals with estimates of fewer than one copy per genome were founder animals, suggesting somatic mosaicism.

FIG. 6

Pedigree analysis of mice generated from two independent founder mice reveals that in both cases, BAC transgenes have inserted in two separate, segregating locations in the genome as demonstrated by number estimates. In both cases this was supported by ~75% rate of transgenesis in F1 progeny (non-transgenic littermates not shown). Copy number estimates for individuals are shown in red. Inset images show representative XGal stained e12.5 embryos characteristic of each independent integration event. (a) 5’ Bmp4 BAC Line 1 founder female generated mice with “high” (Avg. = 9.5) and “low” (Avg. = 3.7) copy number estimates that segregate independently. For the founder, copy number estimates were based on the average of two independent tail biopsies/DNA preps (*). (b) 3’ Bmp4 BAC Line 45 founder female generated F1 progeny with “high” (Avg. = >48) and “low” (Avg. = 2.1) copy number estimates that segregate independently.

FIG. 7

XGal stained embryos generated from three distinct BAC transgene constructs suggest that increasing BAC transgene copy numbers correlate with increased transgene expression. Each row of images represents embryos from a separate BAC construct, arranged by increasing BAC copy number estimates. Top row: e15.5 embryos from independent Bmp4 3’BAC lines. Middle row: e12.5 embryos from independent Bmp4 5’BAC lines. Bottom row: e15.5 Bmp2 deletion-BAC transgenic founder embryos.

FIG. 8

Polymorphic marker analysis suggests that transgenic lines that have multiple BAC copies are likely to carry some intact BAC molecules. Polymorphic markers denoted along the length of Bmp4 5’BAC (top right) and Bmp4 3’BAC (top left; scale bar=20kb). Shown below each BAC are schematics representing lines for which all BAC markers are present, suggesting intact BAC transgenes (solid bars), and lines containing fragmented BAC transgenes (interrupted bars). Solid lines indicate presence of contiguous transgene-specific markers. Open regions indicate loss of transgene-specific markers, and hatched regions indicate regions of potential breakpoints.

FIG. 9

Southern blot analysis on high molecular weight DNA samples from low copy (5’ L12, avg. copy number = 2) and high copy (5’ L1a, avg. copy number = 11) Bmp4 BAC lines suggests intact transgene copies. (a) Image of ethidium bromide stained pulsed-field gel following electrophoresis of agarose-embedded and MluI-digested high molecular weight DNAs, isolated from embryos generated from 5’ BAC carrying stables lines (see Methods). Also included are control digestions of purified 5’ BAC DNAs (50 ng DNA per lane). (b) Phosphor-image of gel shown in A following Southern transfer and hybridization with radiolabeled probe (IRES-β-geo cassette). The ~110 kb doublet (asterisk) in blot lane 1 represents the expected MluI fragments from purified (unmethylated) 5’ BAC DNA. In the lanes with transgenic mouse DNA digested with MluI, bands are evident (arrowhead) that are approximately the full-length size of the 5’ BAC transgenes (~235 kb)(note, the high copy line yields stronger bands than the low copy line). This strongly suggests that one or more copies of transgenes are intact in both the high and low copy 5’ BAC lines.