Assessment of Gene Copy Numbers Using Quantitative Real-time PCR

Quantitative real-time PCRs (qPCR) of genomic DNA were performed to verify results from FISH analyses and to identify potential hPARP-1 homozygotes within the group of mutant mice that were obtained by interbreeding of hPARP-1 hemizygotes (section 4.1.1.4). Further studies were carried out to analyze if gene copy numbers of mParp-1-flanking genes were affected by the gene targeting.

4.1.2.2.1 Validation of Quantitative Real-time PCR

Gene copy numbers of four different genes were analyzed by qPCR: mParp-1, hPARP-1, Gm821 (proximal of mParp-1), and Lin9 (distal of mParp-1). Murine Parp-1 gene copy numbers were analyzed using an approach with mParp-1- and actin-specific Taq-Man-like probes. Amplification of the actin sequence was used as a DNA standard. PCR was set up and validated by J. Diefenbach (formerly University of Konstanz, data not shown). A Sybr Green approach was chosen to determine gene copy numbers of hPARP-1, Gm821, and Lin9 (Figure 4.7 A). Amplification of a highly conserved region of Cytoglobin b (Cygb) with identical primer binding sites for human and murine sequences was used as a DNA standard. When analyzing dilution series of genomic DNA by qPCRs for hPARP-1, Cygb, Gm821, Lin9, strong correlations were obtained between the used amount of DNA (10-200 ng) and the calculated relative gene copy numbers (2^-ΔΔCt values, Figure 4.7 A). Moreover, melt curve analyses confirmed the formation of a single amplicon in the case of each reaction, thereby verifying the specificity of the reactions (Figure 3.2). Quantitative PCR for hPARP-1 gene copy numbers was additionally validated by crossing potentially homozygous hPARP-1 mice (n=4) with wild-type mice and subsequent genotyping of the obtained litters. Figure 4.7 B shows that mice identified as homozygous for hPARP-1 by qPCR (section 4.1.2.2.2) produced litters comprising 100%

mutant mice which is to be expected in case of parental homozygosity.

Figure 4.7. Validation of quantitative real-time PCR.

A. Quantitative real-time PCR (qPCR) specific for hPARP-1, Cygb, Lin9, and Gm821. PCRs were conducted with dilution series of 10-200 ng of genomic DNA isolated from a hemizygous hPARP-1 mouse (hPARP-1 and Cygb) or a wild-type (wt) mouse (Lin9 and Gm821). Samples with 200 ng DNA were used as a standard and 2^-ΔΔCt values of these samples were set as 1.0. Means ± SEM, n=3. B. Validation of results from qPCR by test breeding and genotyping PCR. For this, four homozygous hPARP-1 mice of lines #113NeoR- and #225NeoR- were identified by qPCR and mated with wild-type C57BL/6 mice. As expected by Mendelian law, offspring („2×hPARP-1’ × B6) was 100% mutant (2 representative litters out of 5). ESC indicates embryonic stem cells; f, female; H2O, water control; m, male; NeoR-, lines with excised NeoR cassette.

4.1.2.2.2 Analyses of PARP-1 Gene Copy Numbers and Identification of hPARP-1 Homozygotes

For the analysis of hPARP-1 gene copy numbers, copy numbers of the human diploid fibroblast cell line IMR90 was set as 2.0. Consistent with the results from breeding data and FISH analyses, but prima facie not in accordance with the results from flanking PCR and Southern blot analyses, the presence of two gene copies of mParp-1 and one copy of hPARP-1 was detected in the genome of ES cell clones #113, #225, and #267 (Figure 4.8 A and B). As expected, fully hemizygous hPARP-1 mice of F1 (lines #113 and #225) showed one gene copy of hPARP-1 (Figure 4.8 B). Importantly, hPARP-1 homozygotes carrying two copies of hPARP-1 (lines #113 and #225) were identified in litters that were obtained by intercrossing of hPARP-1 hemizygotes (Figure 4.8 B). These findings were confirmed in the case of line #225 via the analysis of Southern blot signal intensities (section 4.2.1.1, Figure 4.11).

Determination of gene copy numbers of 19 litters (145 mice) revealed a distribution of genotypes [wild-type, hemizygous mutant (1×hPARP-1), and homozygous mutant (2×hPARP-1)] which nearly fits the expected Mendelian distribution (Table 4.3; compare to Table 4.4), even though hPARP-1 hemizygotes were slightly underrepresented.

Table 4.3. Genotype distribution including gene copy numbers of mice of line #225 (F2).

Genotype Offspring Expected

Wild-type 38 (26%) 25%

Hemizygous (1×hPARP-1) 62 (43%) 50%

Homozygous (2×hPARP-1) 45 (31%) 25%

P value (Chi-square test) 0.16

The cohort was obtained by intercrossing of hPARP-1 hemizygotes of F1. 19 litters analyzed, mean litter size: 7.6 animals. Chi square test was performed to compare the observed genotype ratio with the expected ratio. F1/2 indicates first/second filial generation.

Together, data obtained from animal breeding, FISH, and qPCR strongly indicate that a so-called “ectopic gene targeting event” (Dellaire et al. 1997) occurred in the three independently generated ES cell clones, #113, #225, #267. Such an event can be explained as a gene conversion, in which the hPARP-1 targeting vector homology arms invaded the endogenous mParp-1 locus at the promoter and the terminator sites, copied sequences onto their ends, and the vector then integrated at an adjacent non-homologous position with the endogenous mouse locus still present (Figure 5.1). Hence, the flanking PCR and Southern blot analyses registered this as a bona fide targeted recombination.

Figure 4.8. Gene copy numbers of hPARP-1 and mParp-1 in ES cells and mice.

A. Gene copy numbers (GCNs) of mParp-1 as determined by quantitative real-time PCR (qPCR) of genomic DNA from ES cell clones (ESC) and mouse tail biopsies. Murine wild-type (wt) ES cells were used as a standard and mParp-1 GCN was set as 2.0. B. Gene copy numbers of hPARP-1 as determined by qPCR. Human diploid fibroblasts (IMR90) served as a standard with hPARP-1 GCN set as 2.0. Means ± SEM, n=3. Cont. indicates control; F1/2, first/ second filial generation; f, female; m, male.

Figure 4.9. Determination of gene copy numbers of mParp-1 flanking genes.

A. Detail from NCBI Map Viewer showing flanking gene loci of mParp-1 on chromosome 1. B. Genomic DNA from wild-type (wt) mice and hPARP-1 hemizygotes of line #113NeoR- and #225NeoR- were analyzed for hPARP-1, Gm821, and Lin9 gene copy numbers by quantitative PCR. Gm821 and Lin9 flank the mParp-1 gene on its proximal or distal side, respectively. Gm821 and Lin9 gene copy numbers of wild-type mice were used as a standard and set as 2.0. Means ± SEM, n=3. F indicates female; m, male.

4.1.2.2.3 Analysis of Murine Parp-1-flanking Genes

Possible gene duplications of the mParp-1-flanking genes Gm821 (proximal) and Lin9 (distal) were investigated (Figure 4.9 A) to prove potential ectopic gene targeting events in ES cell clones #113, #225, and #267. Murine wild-type DNA was used as a standard, setting gene copy

numbers as 2.0. Quantitative PCR analyses revealed that hPARP-1 mice of lines #113 and #225 carried duplicated regions of Gm821, which corresponds to three copies in hPARP-1 hemizygotes. In addition, line #225 presumably carried a duplication of Lin9 (Figure 4.9 B).

In conclusion, each of the targeted murine chromosomes 1 of ES cell clones #113, #225, and

#267 carried one copy of the endogenous mParp-1 plus one ectopic copy of the hPARP-1.

Presumably, this genetic situation was obtained by a mechanism related to a phenomenon that was formerly described with „ends-in‟ integration vectors in non-ES-cell gene targeting as

„ectopic gene targeting‟ (Dellaire et al. 1997; McCulloch et al. 2003).

4.1.3 Excision of the Neomycin Resistance Cassette

A neomycin resistance (NeoR) cassette flanked by loxP sites was inserted within intron 14 of the hPARP-1 coding sequence to enable positive selection of the targeted ES cells with the antibiotic G418. It was reported that transcription of the NeoR cassette can interfere with the transcription of the gene of interest leading to lower expression levels (Nagy 2000; Nagy 2003).

For this reason the Cre/loxP system was employed to excise the NeoR cassette in hPARP-1 ES cells and mice (Figure 4.10 A and B).