Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay. Results The cell line bore a missense mutation in the 6 coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

cell lines: 50 % in MCF10A-7SD8 and 30 % in MCF10A-ST1 comparing to MCF-7, and little in MCF-10A by wounded healing. These results indicate that Caveolin-1 could significantly mediate membrane-initiated estrogen-signaling pathway, and may play an important role in mammary tumorigensis and may have effect on cell metastasis in vitro. Such evidence will greatly advance the progress in prevention and treatment of human breast cancer.

Lung morphogenesis is a highly regulated process that could be impaired by nutrition deficiency and recent research suggests that disturbed early lung development may pave the way for later illness and accelerated senescence. Vitamin A (retinol) and their derivatives (retinoic acids, RA) are known key developmental regulators that bind and activate retinoic acid receptors (RARs). To evaluate whether marginal vitamin A deficiency (MVAD) begun from pregnancy alters the lung structure and extracellular matrix, we monitored lung morphology, collagen and elastin fiber at postnatal day 1, week 2 and adulthood (week 8) on MVAD and control group. In addition to morphological change, lung RA receptor (RARa, ß, and g) expression was analyzed by immunofluorescence, whereas mRNA levels were measured using RT-PCR. We demonstrate that MVAD begun from pregnancy resulted in lower lung weight, reduced numbers of alveoli and total alveolar surface area, in addition to increased alveoli septa thickness till to adulthood. Exposure to MVAD also resulted in increased collagen deposits and decreasing elastin fiber at postnatal week 2 and 8 in an unorganized manner. Over the normal course of development, total protein and mRNA levels for the RARs declined, but immunofluorescence and RT-PCR demonstrate that exposure to MVAD during the pregnancy period resulted in an immediate and durative increase in RARs levels from postnatal day 1 to adulthood, especially at postnatal week 2. In summary, this study demonstrates that developing lungs are sensitive to MVAD and this effect is permanent throughout the life of the animal and may be mediated in part through augmentation of transcriptional signals in the retinoid pathway. Thus, we hypothesize that durative pregnancy MVAD could impact lung development and result in the permanent impairment which may underlie at least some susceptibility to adult-onset chronic lung disease.Due to the growing concerns over the toxicity and immumogenicity of viral gene delivery systems, gene delivery via nonviral routes has become more desirable and advantageous. In this work, we developed a self-assembled nonviral gene carrier, glutamic acid modified magnetic nanoparticles (GMMN), which were formed by modifying glutamic acid to the surface of Znfe 2 O 4 nanoparticles. Transmission electromicroscopy results indicated that these particles were 30 nm and below in diameter with a narrow size distribution. Zeta potential demonstrated that the GMCN had 12.8 mV positive surface charges due to the exposed amino groups outward on the surface of magnetic nanoparticles. Gel retardation assay and co-sedimentation assay showed a high affinity of GMMN for DNA under physiological conditions. The DNA encapsuled inside the GMMN was protected from the external DNAase environment. The cell culture experiments showed that the GMMN were internalized into human hepatocyte QSG7701 cells and exhibited higher efficiency of intracellular uptake than bared magnetic nanoparticles. Furthermore, the GMMN-DNA complex had no obvious cytotoxici...