Echocardiography and H&E staining.

<p>TG mice and their NTG littermates were analyzed using M-mode echocardiographic analyses at 1, 3, 5, and 7 months of age. Fractional shortening (FS%) was decreased by the expression of HB-EGF compared with that of NTG mice (A). The left ventricular end-systolic diameter (LVIDs) was increased by the transgenic expression of HB-EGF compared with that of NTG mice (B), while left ventricular anterior wall thickness at end-systole (LVAWs) was not different (C). The whole heart (original magnification ×4) from TG and NTG littermates were sampled and stained using H&E and observed with a microscope. Cardiomyocytes (original magnification ×400) from the same areas of the left ventricle from TG and NTG littermates were compared to detect changes of myocyte cross-section size (D). About 50 cells per sample slice were randomly selected to calculate the average area of a single cell size (E, <i>n</i> = 4). *<i>P</i><0.05 <i>vs.</i> NTG mice; <i>P</i><0.001 <i>vs.</i> NTG mice.</p

HB-EGF induced proliferation of cultured primary cardiac fibroblasts and synthesis of collagen <i>in vitro</i>.

<p>Cultured primary cardiac fibroblasts were treated with HB-EGF at various time points. Phosphorylation of Akt, mTor and p70s6k was analyzed using western immunoblot (A). The cultured primary cardiac fibroblasts were treated with DMSO (vehicle control), HB-EGF, HB-EGF + Rapa, and HB-EGF +CRM197. Expression of collagen I was then detected after treatment for 72 hr and phosphorylation of mTor was detected after treatment for 2 hr using western immunoblot (B). The cell proliferation was detected using BrdU staining and the BrdU-positive cells were counted from six sections (C, D). *<i>P</i><0.05 <i>vs.</i> NTG mice; **<i>P</i><0.001 <i>vs.</i> NTG mice.</p

Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

<div><p>The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (<i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i>) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the off-target mutagenesis and mosaicism are rarely detected by comprehensive analysis. Thus, the CRISPR/Cas9 system makes it possible to efficiently and reliably generate gene knock-out rats.</p></div

Heparin-Binding EGF-Like Growth Factor Induces Heart Interstitial Fibrosis via an Akt/mTor/p70s6k Pathway

<div><p>Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is essential for maintaining normal function of the adult heart and is known to play an important role in myocardial remodeling. In the present study, we observed that heart-specific HB-EGF transgenic (TG) mice had systolic dysfunction with decreased fractional shortening (FS%), increased end-systolic diameter (LVIDs) at 5 months of age, increased heart fibrosis, and increased mRNA expression of Col1α1 and Col3α1 at 1, 3, 5 and 7 months of age compared to nontransgenic (NTG) littermates. However, the left ventricular anterior wall thickness at end-systole (LVAWs) of the TG mice was not different than the NTG mice. Phosphorylation levels of Akt, mTor and p70s6k were increased due to HB-EGF expression in TG mice compared with the NTG mice at 3 and 7 months of age. Additionally, activated Akt, mTor and p70s6k were co-localized with vimentin to cardiac fibroblasts isolated from TG mice. Furthermore, HB-EGF significantly increased phosphorylation levels of Akt, mTor and p70s6k and increased expression of type I collagen in cultured primary cardiac fibroblasts. Rapamycin (Rapa) and CRM197, inhibitors of mTor and HB-EGF respectively, could inhibit the expression of type I collagen in the cultured primary cardiac fibroblasts and Rapa suppressed interstitial fibrosis of the heart tissues <em>in vivo</em>. In addition, a BrdU assay showed that HB-EGF increased proliferation of cardiac fibroblasts by 30% compared with cells without HB-EGF treatment. HB-EGF-induced proliferation was completely diminished in the presence of Rapa. These results suggest that HB-EGF induced heart fibrosis and proliferation of cardiac fibroblasts occurs through activation of the Akt/mTor/p70s6k pathway.</p> </div

Summary of the mutation of the founder rats.

<p>Single sgRNA/Gene: A mixture of 4 sgRNAs, each targeting a single site in each of the 4 genes. Dual sgRNAs/Gene: Another distinct sgRNA of each gene, together with the tested 4 sgRNAs targeting double site in each of the 4 genes.</p

Generation of multiplex genetic modified rats using CRISPR/Cas9 system.

<p>(a) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> by T7EN1 cleavage assay. PCR amplicon of the targeted fragment at the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in 15 founder rats (#1∼15) were subjected to T7EN1 cleavage assay. Founder #13, which is quadruple gene mutant, was marked with asterisks. (b) DNA sequences of four loci in founder #13. PCR amplicon with cleaved bands in T7EN1 cleavage assay were cloned and sequenced. The PAM sequence was underlined and highlighted in green; the targeting site are red; the mutations are blue, lower case; insertions (+) or deletions (−) are shown to the right of each allele.</p

Cas9:sgRNA-mediated modifications in 4 genes by a mixture of dual sgRNAs for each gene.

<p>(a) PCR identification of sgRNA:Cas9-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in 25 founder rats (#16∼40) derived from co-microinjection of a mixture of dual sgRNAs for each genes as described in Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089413#pone.0089413.s001" target="_blank">File S1</a>. Primer used for PCR amplication was described in Table S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089413#pone.0089413.s001" target="_blank">File S1</a>. Additionally, founder #36, #38 had a larger deletion in the <i>ApoE</i>, the primer <i>ApoE</i>-NS2 and <i>ApoE</i>-NAS2 used for amplification. Founder #38 had a larger deletion in the <i>B2m</i>, the primer <i>B2m</i>-S2 and <i>B2m</i>-AS2 used for amplification. (b) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> by T7EN1 cleavage assay. PCR products from (a) were subjected to T7EN1 cleavage assay as described in material and methods.</p

Determination of the phosphorylation levels of Akt, mTor and p70s6k in heart tissues.

<p>Mice were sacrificed at 3 and 7 months of age, and heart-tissue samples were homogenized in RIPA buffer. Phosphorylation levels of Akt, mTor and p70s6k were detected using western immunoblot (A). The relative intensities were detected using Image J and compared with those of NTG mice (B). *<i>P</i><0.05 <i>vs.</i> NTG mice at 3month; **<i>P</i><0.001 <i>vs.</i> NTG mice at 3month; †<i>P</i><0.05 <i>vs.</i> NTG mice at 7month; ‡<i>P</i><0.001 <i>vs.</i> NTG mice at 7month.</p

Localization of activated Akt, mTor and p70s6k in mouse heart tissues using double immunofluorescence confocal staining at 7 months of age.

<p>Cell localization of p-Akt (A), p-mTor (B) and p-p70s6k (C) were observed using confocal laser- scanning microscopy (Leica TCS SP2, Germany). Scale bar = 20 µm.</p

Generation of TG mice.

<p>The HB-EGF TG construct was generated by the insertion of a target gene under the control of the α-MHC cardiac-specific promoter (A), and the TG mice were created using a microinjection method. Mouse lines with overexpression of HB-EGF (proHB-EGF and mature HB-EGF) were selected using a western immunoblot procedure and normalized with GAPDH (B).</p