Generation of a cre recombinase-conditional Nos1ap over-expression transgenic mouse

Abstract

Polymorphic non-coding variants at the NOS1AP locus have been associated with the common cardiac, metabolic and neurological traits and diseases. Although, in vitro gene targeting-based cellular and biochemical studies have shed some light on NOS1AP function in cardiac and neuronal tissue, to enhance our understanding of NOS1AP function in mammalian physiology and disease, we report the generation of cre recombinase-conditional Nos1ap over-expression transgenic mice (Nos1ap (Tg)). Conditional transgenic mice were generated by the pronuclear injection method and three independent, single-site, multiple copies integration event-based founder lines were selected. For heart-restricted over-expression, Nos1ap (Tg) mice were crossed with Mlc2v-cre and Nos1ap transcript over-expression was observed in left ventricles from Nos1ap (Tg); Mlc2v-cre F1 mice. We believe that with the potential of conditional over-expression, Nos1ap (Tg) mice will be a useful resource in studying NOS1AP function in various tissues under physiological and disease states.

Fig. 1

Generation of cre recombinase-conditional Nos1ap over-expression transgenic mice. a Nos1ap pCLIP transgene construct (adapted from George et al. 2007), and representation for multiple copy head-to-tail insertion of the transgene and the expected 6.8 kb Southern band with lacZ probe (P) and EcoRV (E) digestion; βgeo β-galactosidase-neomycin fusion gene, pA signal, IRES internal ribosome entry site; b identification of transgenic founder by Southern blotting. Southern blotting performed on mouse tail genomic DNA digested with EcoRV and probed using lacZ specific probe. Lanes 3–8 have wild type mouse tail genomic DNA spiked with zero or multiple copies, as indicated, of Nos1ap pCLIP plasmid DNA per diploid genome. Mouse 112 was selected as a founder line for further experiments; c single site transgene integration in cre recombinase-conditional Nos1ap over-expression transgenic lines. Metaphase FISH using Nos1ap pCLIP plasmid specific probe performed in cultured peripheral blood cells of F₁ mice derived from cross between 112, 314, and 333 transgenic founders and FVB mice. Red dots in the left panel (white arrow) indicate site of integration on chromosome 4 (top), chromosome 12 (middle) and chromosome 2 (bottom) for 112, 314 and 333 lines, respectively

Fig. 2

Nos1ap^Tg; Mlc2v-cre (Tg⁺;cre⁺) mice from two transgenic lines overexpress Nos1ap transcript in left ventricles. a Box-and-Whisker plots showing relative expression levels for Nos1ap transcript in left ventricles from Tg⁺;cre⁺ (n = 10), Tg⁺;cre⁻ (conditional transgene only, n = 3) and wild type control littermates (Tg⁻;cre⁻, n = 10) mice derived from transgenic founder 112. Expression of Nos1ap transcript was higher in Tg⁺;cre⁺ mice as compared to Tg⁻;cre⁻ mice, t(18) = 5.77, p <0.001 and expression of Nos1ap transcript was not significantly different between Tg⁺;cre⁻ and Tg⁻;cre⁻mice, t(11) = 0.66, p = 0.52; b same as a, mice derived from transgenic founder 333 (Tg⁺;cre⁺ n = 7, Tg⁺;cre⁻ n = 5, Tg⁻;cre⁻ n = 7). Expression of Nos1ap transcript was higher in Tg⁺;cre⁺ mice as compared to Tg⁻;cre⁻ mice, t(12) = 5.98, p <0.001 and expression of Nos1ap transcript was not significantly different between Tg⁺;cre⁻ and Tg⁻;cre⁻ mice, t(10) = 0.07, p = 0.94; c Same as a, mice derived from transgenic founder 314 (Tg⁺;cre⁺ n = 8, Tg⁻;cre⁻ n = 7). No significant difference in expression level was observed between the two groups of mice derived from transgenic founder 314, t(13) = 0.22, p = 0.83