Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.

CD80CD86 deficiency disrupts regulatory CD4+FoxP3+T cell homoeostasis and induces autoimmune-like alopecia.

Alopecia areata (AA) is an autoimmune disease that results in spot baldness in humans. Adequate animal models for AA are currently lacking. The objective of this study was to elucidate the mechanism of autoimmune-like alopecia (ALA) in C57BL/6.CD80CD86-deficient (B6.CD80CD86-/- ) mice. Incidence and severity of alopecia were analysed in 58 B6.CD80CD86-/- mice using histological examination, flow cytometry, multiplex enzyme-linked immunosorbent assay, quantitative RT-PCR and CD25 inhibition test. Both male and female B6.CD80CD86-/- mice showed almost 100% incidence of hair loss at 40 weeks of age. Moreover, CD4+FoxP3+Treg (Treg) cell population in B6.CD80CD86-/- mice was significantly lower than in B6 mice, which presumably underlined autoimmune reaction. Histologically, B6.CD80CD86-/- mice showed CD4+ and CD8+ T-cell infiltration around terminal follicle region and exhibited hair follicle destruction in the anagen or catagen stage. Negative correlation between the number of CD4+FoxP3+ Tregs and ALA was confirmed by the CD25 depletion test in B6 mice, as follicle destruction was similar to that observed in B6.CD80CD86-/- animals. CD80CD86 deficiency disrupted CD4+FoxP3+ Treg homoeostasis and prompted the development of ALA. We demonstrated that B6.CD80CD86-/- mice might have several advantages as an ALA model, because they exhibited high incidence of disease phenotype and epipathogenesis similar to that observed in human AA.

5-Bromo-2'-deoxyuridine induced effluvium via p53-mediated CD326-positive keratinocyte apoptosis in C57BL/6 mice.

Anagen effluvium develops because of disturbances in the hair follicle cycle, leading to acute and severe hair loss in humans. The objective of this study was to establish a mouse model of anagen effluvium by 5-bromo-2'-deoxyuridine (BrdU) treatment, and evaluate the pathological changes and underlying mechanisms. We treated 9-10-day-old pups and 3-7-week-old C57BL/6 mice with BrdU. After successfully inducing hair loss in the neonatal pups, microscopic, immunohistochemical and flow cytometry analyses were conducted. BrdU induced early onset alopecia in neonates and caused epidermal thickening and hair shaft breakage. BrdU appeared to incorporate the CD326-positive keratinocyte layer and induced p53-related apoptosis. Keratinocyte apoptosis caused immune cell infiltration in the dermal region; M2 macrophages and neutrophils were dominant. The BrdU-induced hair loss was dose-dependent, and alopecia was visible at a dose range of 25-200 µg/g bodyweight. The BrdU-induced anagen effluvium mouse model is novel and easily established by administrating four simple BrdU injections to pups; these mice showed synchronized onset of alopecia symptoms with little individual variation. Moreover, this model showed an alopecia phenotype similar to that of human anagen effluvium with acute, severe and widespread hair loss.

Premature mammary gland involution with repeated corticosterone injection in interleukin 10-deficient mice.

Recently, we found that maternal stress could induce premature mammary gland involution in interleukin 10 knock out (IL-10-/-) mice. To elucidate correlation between stress, IL-10, and mammary gland involution, corticosterone was injected into the lactating wild type and IL-10-deficient mice and assessed mammary gland phenotype. Repetitive corticosterone injection developed premature mammary gland involution only in B6.IL-10-/- mice; moreover, it induced alopecia in nursing pups. Corticosterone injection induced several typical changes such as mammary gland epithelial cell apoptosis, macrophage infiltration, fat deposition in adipocyte, STAT3 phosphorylation, and upregulation of tyrosine hydroxylase gene in adrenal gland. Overall incidence of pup alopecia and mammary gland involution was relatively high in corticosterone than control B6.IL-10-/- group (57% vs. 20%). Our finding demonstrates that IL-10 is important for stress modulation, and B6.Il-10-/- with corticosterone has several advantage such as simple to establish, well-defined onset of mammary gland involution, high incidence, and inducing pup alopecia.

Pregnancy in postpartum estrus induces inflammatory milk production and catagen specific pup skin inflammation in interleukin-10 deficient mice.

BACKGROUND: The interleukin 10 deficient mice (IL-10(-/-)) showed high incidence of pup alopecia compared to other strains, and pup alopecia was caused by skin inflammation and was recoverable. Pup alopecia of B6.IL-10(-/-) might be related with maternal factor and interleukin-10 deficient phenotype. OBJECTIVE: The objectives of this study were elucidating of maternal factors for inflammatory milk production and characterization of pup alopecia in IL-10(-/-) mice. METHODS: Incidences of pup alopecia were analyzed with 13 breeding cases. Comparison between control and alopecia pups and its dams, were conducted with histological examination (H&E, TUNEL assay, immunohistochemistry for F4/80, iNOS, CD206, Gr-1, CD4, CD8, CD11c and CD326), fostering test, forced weaning test, qPCR for tyrosine hydroxylase, flow cytometry, IL-10 inhibition test, BMDM stimulation test and LC/MS analysis. RESULTS: Presence of pregnancy in postpartum estrus showed significant correlation with inflammatory milk production and mammary gland involution in B6.IL-10(-/-) mice. There were no different mass in inflammatory milk, but different ionization intensity was detected. Inflammatory milk directly induced hepatocyte steatosis, catagen stage specific hair breaking and alopeicia in pups. Histologically, hypertropy of outer root sheath and macrophage/neutrophil infiltration were typical. CONCLUSION: B6.IL-10(-/-) dam with stress such as PPE could produce untimely mammary gland involution and inflammatory milk production. Interleukin 10 is important for maternal stress regulation and protecting inflammatory milk production, also influence severity of pup skin inflammation and alopecia. Remarkably, inflammatory milk induced hepatocyte steatosis, and it could indicate there is abnormal lipid metabolism. This was first report for catagen specific alopecia in mouse.

Size-dependent plasmonic responses of single gold nanoparticles for analysis of biorecognition.

We report the use of plasmonic responses of single gold nanoparticles (AuNPs) with various sizes for the analysis of biomolecular recognition. We also describe the relationship between particle size and plasmonic response induced by the binding of receptors and target analytes. To investigate the plasmonic response of AuNPs, Rayleigh light scattering spectra were collected from individual AuNPs using a dark-field microspectroscopy system. Using prostate-specific antigen (PSA) as a model, the linear dynamic range was obtained in the concentration range of 10(-4) to 10 ng/ml, with the smallest detectable concentration at 0.1 pg/ml corresponding to localized surface plasmon resonance (LSPR) λ(max) shifts of approximately 2.95 nm. This result indicates that individual AuNPs can be used for development of a very sensitive, robust, simple, and label-free biosensor to detect protein biomarkers. Furthermore, the method possesses great potential for monitoring other biological interactions.

A strategy for the ultrasensitive detection of cancer biomarkers based on the LSPR response of a single AuNP.

A biosensor based on the localized surface plasmon resonance (LSPR) response of a single Au nanoparticle was fabricated for the highly sensitive detection and quantification of a specific cancer biomarker. The spectral position changes of single Au nanoparticles induced by the binding of adsorbates and target analytes were effectively utilized as sensing tools. The LSPR responses of single Au nanoparticles were obtained by tracking the wavelength shift of the corresponding resonant Rayleigh light scattering spectra via dark-field microspectroscopy. Using prostate specific antigen as a model, an LSPR lambda max shift of about 2.75 nm was recorded by a primary immunoresponse corresponding to 0.1 pg/mL of the target antigen. The sensitivity of the immunoassay can be substantially enhanced, however, by a sandwich strategy. A PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, the linear dynamic range of the sensing platform was determined to be within the concentration range of 10(-4) to 0.1 ng/mL and a detectable minimum concentration of 0.1 pg/mL was identified, with an LSPR lambda max shift of about 4.96 nm. The results indicate that the aforementioned approach can significantly contribute to the fabrication of ultrasensitive biosensors, allowing the quantitative analysis of cancer-associated proteins