Assessment of Artificial MiRNA Architectures for Higher Knockdown Efficiencies without the Undesired Effects in Mice

Abstract

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both "-MG amiRNA" cells and "amiRNA low-expression" cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs.

Fig 1. Comparison of knockdown efficiencies of different amiR-eGFP architectures in eGFP-expressing ES cells.

(A) Structures of amiR-eGFP expression vectors. Two amiRNAs, each of which was designed to target different positions of the eGFP gene (hairpin structures in pink, for eGFP123, and blue, for eGFP419), were used (See S1 Table for details). (B–D) Fluorescence intensity determined after transfecting eGFP-expressing ES cells with each vector (No. 1 to 6 in A). At 2 days after transfection, the fluorescence intensity of eGFP (B and C) and tdTomato (D) was assessed by FACS. Orange lines (arrowheads in B and C) are eGFP fluorescence intensities of non-transformed eGFP-expressing ES cells (control: C). The mean fluorescence intensities (MFIs) are indicated in the upper right-hand corner of each graph in (B–D). Numbers 1 to 6 in (B–D) correspond to those shown in (A).

Fig 2. Removing the tdTomato marker gene from an amiRNA expression cassette enhances knockdown efficiency.

(A) Schematic representation of the experimental strategy. An amiR-eGFP expression vector (pPBKD-G1) that contained a tdTomato marker gene flanked by rox sites was co-transfected into eGFP-expressing ES cells along with a pPBase and a pPBN. Stably transformed clones that exhibited tdTomato fluorescence (+MG clone) were isolated first. Then, these clones were administrated Dre recombinase to remove the tdTomato gene, which resulted in generating a marker-less clone (−MG clone). (B) eGFP fluorescence intensity in ES cells. The experiments were repeated six times and the representative histogram is shown (left). Cell lines 3 (solid line) and 4 (dot line) were used for analysis. Red and blue lines represent eGFP fluorescence in a “+MG clone” and a “−MG clone,” respectively. Green and black lines indicate eGFP fluorescence intensity in eGFP-expressing and wild-type (eGFP-negative) ES cells, respectively. The box plots show the normalized MFI obtained in six experiments (right); *P = 1.8 × 10⁻³, **P = 1.4 × 10⁻⁵ (paired t-test). (C) Quantitative real-time PCR of amiR-eGFP123 (left) and amiR-eGFP419 (right) in cell lines 3 (+MG and −MG) and 4 (+MG and −MG); The experiments were repeated three times; *P = 2.6 × 10⁻³, **P = 6.9 × 10⁻³ (two tailed Student’s t-test). The differences are not significant in amiR-eGFP419 expression (P > 0.05).

Fig 3. Knockdown efficiency in AWV^Δex mice.

(A) eGFP and tdTomato fluorescent signals in mouse organs. Mouse genotypes (No. 1 to 7) are indicated on the right. Mice 5 to 7 are AWV^Δex/eGFP mice, in which the transgene included in each mouse had been derived from a different founder (F0) mouse (lines 1 to 3). An amiRNA expression cassette in AWV^Δex Tg mice is shown (upper right). (B) eGFP fluorescent signals in spleen cells analyzed by FACS. (C) Quantitative real-time PCR of eGFP mRNA expression in mouse liver and kidney tissues. (D) tdTomato fluorescent signals in spleen cells analyzed by FACS. Numbers 1 to 7 in (B–D) correspond to those shown in (A).

Fig 4. Generation of marker gene-less (−MG) amiR-eGFP-expressing mice.

(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 in vitro 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 S5 Fig. (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.