45 research outputs found
Rational Design of Mini-Cas9 for Transcriptional Activation
Nuclease dead Cas9 (dCas9) has been
widely used for modulating
gene expression by fusing with different activation or repression
domains. However, delivery of the CRISPR/Cas system fused with various
effector domains in a single adeno-associated virus (AAV) remains
challenging due to the payload limit. Here, we engineered a set of
downsized variants of Cas9 including <i>Staphylococcus aureus</i> Cas9 (SaCas9) that retained DNA binding activity by deleting conserved
functional domains. We demonstrated that fusing FokI nuclease domain
to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle
of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition,
we constructed a set of compact transactivation domains based on the
tripartite VPR activation domain and self-assembled arrays of split
SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9.
Lastly, we produced a single AAV containing the mini-SaCas9 fused
with a downsized transactivation domain along with an optimized gRNA
expression cassette, which showed efficient transactivation activity.
Our results highlighted a practical approach to generate down-sized
CRISPR/Cas9 and gene activation systems for <i>in vivo</i> applications
Solutions for different concentration of HCO<sub>3</sub><sup>−</sup>.
<p>Solutions for different concentration of HCO<sub>3</sub><sup>−</sup>.</p
Involvement of CFTR and CAII in bacterial killing <i>in vitro</i>.
<p>(A)When 1×10<sup>4</sup> CFU of <i>E.coli</i> was inoculated to the apical compartment of the rat prostate epithelial cells for 18 h, there was no bacterial activity detected in the culture medium. 10 µM CFTR<sub>inh</sub>-172 (A), 1∶500 CFTR antibody (B) or 50 µM acetazolamide (C) were added with 1×10<sup>5 </sup><i>E.coli</i> to block CFTR or CAII activity and their effect on bacterial activity 18 hours after incubation was shown. (**P<0.01, ***P<0.001).</p
Expression of CFTR and CAII in human hyperplasia prostate with inflammation.
<p>CFTR (A, B) and CAII (C, D) were detected in human hyperplasia tissues. There was lymphocytes infiltration (Yellow arrow, B, D) in the inflamed area of the clinical prostate hyperplasia samples. Note that the expression of CFTR and CAII was stronger in the area with lymphocytes infiltration (Red arrow, B, D) than those without infiltration (Red arrow, A, C). (E) Negative control. Scale bar: 50 µm.</p
Bacterial killing effect of CFTR <i>in vivo</i> and upregulation of cytokines, CFTR and CAII in <i>E coli</i>-infected rat prostate.
<p>(A) Comparison of <i>E coli</i> bacterial activities recovered from rat prostatitis models without or with CFTR<sub>inh</sub>-172 (10 µM). Each point indicates the bacterial CFU per gram of prostate tissue weight (***P<0.001). (B) <i>E.coli</i> up-regulated the expression of cytokine genes, CFTR and CAII in rat prostate as determined by RT-PCR. Data were from three experiments. (C) Expression of CFTR (160 kD) and CAII (29 kD) protein was significantly up-regulated in <i>E.coli</i> -infected rat prostate as determined by western blot. Data were from three experiments. (*P<0.05, **P<0.01, ***P<0.001).</p
HCO<sub>3</sub><sup>−</sup> but not pH exhibits bactericidal capacity <i>in vitro</i>.
<p>(A). The activity of <i>E.coli</i> was inhibited by 80 mM HCO<sub>3</sub><sup>−</sup> and 50 mM HCO<sub>3</sub><sup>−</sup>. (B) Insignificant effect on bacterial activities of varied pH (at constant 25 mM HCO<sub>3</sub><sup>−</sup>) at 7.35, 7.95, 8.14 and 8.24 which was corresponding with the pH value of different concentration of HCO<sub>3</sub><sup>−</sup>. Data were from three experiments. (**P<0.01vs 0 mM HCO<sub>3</sub><sup>−</sup>, <sup>$</sup>p<0.001 vs 25 mM HCO<sub>3</sub><sup>−</sup>,<sup> ##</sup>P<0.01vs 50 mM HCO<sub>3</sub><sup>−</sup>, <sup>&</sup>P<0.05 vs 25 mM HCO<sub>3</sub><sup>−</sup>).</p
Expression of CFTR in rat prostate epithelial cells.
<p>(A) Immunohistochemical staining of CFTR in SD rat prostate with negative control in the absence of primary antibody. CFTR was expressed at the apical surface of rat ventral prostate epithelium. Scale bar: 100 µm. (B) CFTR transcript was detected by RT-PCR in cultured rat prostate epithelial cells with predicted amplification products at 481 bp. (C) CFTR protein was detected in rat prostate epithelial cells by Western blotting which recognizes a band at MW 160 kDa. (D) Phase contrast image (left) and immunofluorescence staining of cytokeratin 5&8 (middle, green) or CFTR (right, green) in rat prostate epithelial cells. Cell nuclei were counterstained with DAPI (blue). Scale bar: 100 µm.</p
LPS-induced upregulation of cytokines, CFTR and CAII expression in rat prostate epithelial cells.
<p>Primary rat prostate epithelial cells were treated with 1 µg/ml <i>E.coli</i>-LPS for 24 h. The expression levels of IL-6, IL-1β, TNF-α, CFTR and CAII were evaluated by RT-PCR (A, B) and GAPDH was used as control. Data were from three experiments. (C) <i>E.coli</i>-LPS up-regulated the protein expression of CFTR (160 kD) and CAII (29 kD)as detected by western blot, with β-tubulin (55 kD) used as the loading control. Data were from three experiments. (*P<0.05, **P<0.01, ***P<0.001).</p
MOESM1 of In vitro chemokine (C-C motif) receptor 6-dependent non-inflammatory chemotaxis during spermatogenesis
Additional file 1: Figure S1. Expression of CCR6 in normal adult human testis. Representative western blot results showing the expression of CCR6 in normal adult human testis (n = 5). β-Tubulin was used as loading control