Percutaneous cerclage wiring for the surgical treatment of displaced patella fractures

The patella plays an important role in the knee joint extension, and a patella fracture requires surgical treatment when it is accompanied by displacement of bone fragments and a joint surface gap. In patella fractures, there is disruption of the soft tissue structures that support the knee extension mechanism. We use a method of percutaneous cerclage wiring to fix the patella and include the peripatellar soft tissues in five patients. All cases were closed fractures, and the AO classification was type A in 1 and type C in 4. At a mean follow-up of 11.2 months, union was achieved in four cases with failure in one inferior pole fracture avulsion. There was no extensor lag noted in any patient, with mean flexion at 141° (120–160). As this percutaneous cerclage wiring method includes soft tissue approximation in the wiring, it may be especially suitable for comminuted fractures for which classic tension band wiring techniques cannot be used. We employed this procedure to atraumatically manipulate peripatellar soft tissues together with the fracture fragments in order to obtain optimal restoration of continuity of the extensor mechanism

CLICK:One-step generation of conditional knockout mice

Abstract Background CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. Results Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. Conclusions The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice

Simple genome editing of rodent intact embryos by electroporation

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

Simple knockout by electroporation of engineered endonucleases into intact rat embryos.

超簡単！遺伝子改変動物作製法の開発 -エレクトロポレーション（電気穿孔）法による哺乳類受精卵への ZFN、TALEN、CRISPR-Casの導入に成功-. 京都大学プレスリリース. 2014-10-03.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

SsODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes

The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms

Development of rat embryos with introduced RNA and ssODN using the TAKE method.

<p>^aCalculated from the number of embryos examined.</p><p>^bCalculated from the number of embryos developed to two cells.</p><p>^cCalculated from the number of male and female rats.</p><p>Significant differences at <i>P</i> < 0.05; a <i>vs</i>. b, c <i>vs</i>. d.</p><p>Development of rat embryos with introduced RNA and ssODN using the TAKE method.</p

Development of mouse embryos with introduced RNAs and ssODN using the TAKE method.

<p>^aCalculated from the number of embryos examined.</p><p>^bCalculated from the number of embryos developed to two cells.</p><p>^cCalculated from the number of male and female mice.</p><p>Significant differences at <i>P</i> < 0.05; a <i>vs</i>. b, c <i>vs</i>. d, e <i>vs</i>. f.</p><p>Development of mouse embryos with introduced RNAs and ssODN using the TAKE method.</p

Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes

TALエフェクターヌクレアーゼ(TALEN)を用いた効率的な遺伝子改変ラットの作製技術. 京都大学プレスリリース. 2013-02-13.TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species

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

新しい人工ヌクレアーゼPlatinum TALENを用いたゲノム編集によって高効率にカエルやラットの遺伝子改変が可能に!. 京都大学プレスリリース. 2013-11-29.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