Generation of marker gene-less (−MG) amiR-eGFP-expressing mice.

<p>(A) Schematic representation of the strategy used to eliminate extra sequences in AWK mice. Extra sequences including vector sequence (shown in moss green line) and an FLPe expression cassette in the ex-allele, were removed by administering FLPe, which resulted in generating an Δex-allele. An FLPe expression cassette in FLPe Tg mice is shown below. Red arrows indicate primer-binding sites used for genotyping analysis. (B) Genotyping results of pups obtained by <i>in vitro</i> fertilization using frozen sperm derived from an AWK^ex/Δex/FLPe mouse (lanes 1 to 20). Lane 21: negative control. PCR conducted using the primer set shown in (A) was used to assess the presence of transgenes. Fifteen individuals (1 to 15) were alive, whereas 5 pups (16 to 20) died just after birth [Note: for the sample No. 20 with primer set (M632/M244), the PCR product was loaded onto a separate lane in the same gel and rearranged in this Figure for clarity]. (C) Body weights of littermates obtained by crossing an AWK^ex mouse with an FLPe Tg mouse. The AWK^ex/Δex/FLPe mouse contained both type of cells (with the AWK^Δex allele or AWK^ex allele) classified as a mosaic mouse. (D) Cataracts developed in the AWK^ex/Δex/FLPe mouse. All mice, similar to those observed in (C), were examined at 66 weeks of age. Cataract phenotypes in additional AWK^ex/Δex/FLPe mice are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135919#pone.0135919.s006" target="_blank">S5 Fig</a>. (E) Scatter plot of quantitative real-time PCR of amiR-eGFP123 expressions (log2 of normalized data) in nine mosaic mice and one AWK^ex mouse including littermates as in (D). Each circle and triangle represents an individual mouse. The values are expressed as mean ± SD represented by the green line for mosaic mice.