Excision of the Neo/Stop sequence in genomic DNA of hPARP-1 transgenic mice

Figure 25: Western blot analysis of hPARP-1 and Cre recombinase expression in lck-Cre x hPARP-1 transgenic mice. Western blot analysis of thymus protein from transgenic mice for hPARP-1 expression (detection with monoclonal antibody FI-23), general PARP-1 expression (detection with monoclonal antibody CII-10) and Cre recombinase expression (detection with polyclonal Cre antibody). β-Actin as loading control. Cre, CO60 cells transfected with turbo-Cre expression plasmid. PC, spleen extracts from an existing hPARP-1 expressing mouse model was used as positive control (Mangerich et al., 2009), obtained from Dr. Aswin Mangerich.

Additionally, hPARP-1 transgenic mice from all founder lines were mated with EIIa-Cre transgenic mice. Their offspring was analyzed for hPARP-1 expression after protein isolation from spleen and kidney (experiments were performed in collaboration with Dr. Aswin Mangerich, University of Konstanz). As shown in a representative Western blot (Figure 26), hPARP-1 protein was not detectable in both organs in any of these transgenic mice, whereas endogenous mouse PARP-1 protein was detectable in the spleen but not in the kidney.

Figure 26: Western blot analysis of PARP-1 expression in kidney and spleen from hPARP-1 x EIIa transgenic mice. Western blot analysis of kidney and spleen from transgenic mice for hPARP-1 expression (detection with monoclonal antibody FI-23) and general PARP-1 expression (detection with monoclonal antibody CII-10). PC, positive control, recombinant hPARP-1 protein, 200 ng. (This experiment was performed in collaboration with Dr. A.

Mangerich, University of Konstanz).

4.3.6 Excision of the Neo/Stop sequence in genomic DNA of hPARP-1 transgenic mice

In order to generate transgenic mice expressing hPARP-1 protein, founder animals which have previously been characterized for successful insertion of the hPARP-1 transgene by

genotyping, were mated with lck-Cre transgenic mice expressing Cre recombinase in T-cells.

Their offspring was expected to show an excision of the transcriptional Neo/Stop sequence in DNA isolated from thymus. Isolated DNA from thymus of those transgenic mice was used for analysis in a flanking PCR reaction. The primers used (50610, 50611) flanking the Neo/Stop sequence, should produce either an amplicon of 3,400 bp in the case of no excision, or an amplicon of 650 bp in the case of successful excision of the Neo/Stop sequence (Figure 27, A). As a result, most of the transgenic mice showed a complete excision of the Neo/Stop sequence on DNA level (Figure 27, B).

Figure 27: Excision of the Neo/Stop sequence after mating of hPARP-1 x lck-Cre tg mice.

(A) Schematic illustration of the excision of the Neo/Stop sequence in vivo by Cre recombinase and the expected length of the amplicons in a PCR reaction before and after successful excision.

(B) Flanking PCR from mouse thymus DNA with successful (3:1m0, tg-2, 2:1f0, tg-4) or incomplete excision (tg-1, tg-3) of the Neo/Stop sequence. Controls: NC, negative control with DNA from wt mouse; P, pUCTE5; Ptrans, transfection of pUCTE5 expression cassette in EL-4 cells; P+Cre, co-transfection of pUCTE5 expression plasmid and turbo-Cre expression plasmid.

tg, transgenic mouse; H2O, water control; M, molecular size ladder; m, male; f, female.

As a control, the correct excision of the Neo/Stop sequence could be confirmed in a cellular assay by using EL-4 cells co-transfected with both pUCTE5 and turbo-Cre (Figure 27, B (P + Cre)).

Figure 28: Excision of the Neo/Stop sequence after mating of hPARP-1 x EIIa-Cre tg mice.

(A) Schematic illustration of the excision of the Neo/Stop sequence in vivo by Cre recombinase and the expected length of amplicon in a PCR reaction before and after successful excision. (B) PCR with DNA of tail biopsies prepared from different mice. Shown are samples of successful (3:1m0, 2:1f0) or incomplete excision (tg mice) of the Neo/Stop sequence. Controls: PC, positive control, lck-Cre x hPARP-1 mouse tail DNA; NC, negative control, wt mouse tail DNA (Experiment was performed in collaboration with Dr. A. Mangerich, University of Konstanz).

In addition, hPARP-1 transgenic mice were mated with ubiquitously Cre recombinase expressing EIIa-Cre mice, the latter mouse type being characterized by expression of Cre recombinase in early embryonic development. This mating should lead to an efficient excision of the Neo/Stop sequence in nearly all cell types of the transgenic offspring derived thereof.

To prove whether the excision was successful, prepared DNA from tail biopsies was analysed by a slightly modified PCR approach depicted in Figure 28, A (experiments were performed in collaboration with Dr. Aswin Mangerich). The chosen primer set (AMa27/28) bind to the neomycin resistance cassette located between the two loxP recognition sites. The PCR reaction should generate an amplicon of 380 bp, if there were cells in which the Neo/Stop sequence was not excised. In case of a complete excision no amplicon is synthesised. To ensure that all offspring mice contained the parental Cre recombinase gene from EIIa-Cre mice, a specific primer set (AMa21/22) for the Cre recombinase gene (408 bp) was used.

Furthermore, a control primer set (AMa25/26) for Fabpi (200 bp, fatty acid binding protein, intestinal) was used to prove whether the PCR reaction had run properly. As depicted in Figure 28, the same hPARP-1 transgenic mice (3:1m0, 2:1f0 from Figure 27, B) previously being mated with lck-Cre mice and showing an excision of the Neo/Stop sequence in the thymus, were also mated with EIIa-Cre mice. Their offspring revealed a complete excision of the Neo/Stop sequence in DNA from tail biopsies (3:1m0, 2:1f0; Figure 28, B). However, in most of the transgenic mice, excision of the Neo/Stop sequence obviously did not occur completely in DNA from tail biopsies, noticeable by a slightly weaker Neo amplicon signal compared with control (Figure 28, B).