Trabajo presentado en el Genomic Instability and DNA Repair, Keystone Symposia, celebrado en Banff, Alberta (Canada) del 3 al 8 de marzo de 2013

Regulation of DNA double strand break repair pathways

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

http://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/215146/1/j.stemcr.2015.01.016.pdf

Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs

Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported However, the functionalities of structurally different TALENs have not yet been compared Here, we report on the functional differences among several types of TALENs targeting the same loci Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering

/pdf/repeating-pattern-of-non-rvd-variations-in-dna-binding-3bebo0xh07.pdf

Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity.

Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.

/pdf/simple-knockout-by-electroporation-of-engineered-3eiuo7z17k.pdf

Simple knockout by electroporation of engineered endonucleases into intact rat embryos

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

/pdf/simple-genome-editing-of-rodent-intact-embryos-by-571dau4h4w.pdf

Simple Genome Editing of Rodent Intact Embryos by Electroporation

http://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/159642/1/j.celrep.2012.08.009.pdf

Generation and characterization of severe combined immunodeficiency rats.

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Generation and characterization of severe combined immunodeficiency rats.