Welcome To Website IAS

Hot news
Achievement

Independence Award

- First Rank - Second Rank - Third Rank

Labour Award

- First Rank - Second Rank -Third Rank

National Award

 - Study on food stuff for animal(2005)

 - Study on rice breeding for export and domestic consumption(2005)

VIFOTEC Award

- Hybrid Maize by Single Cross V2002 (2003)

- Tomato Grafting to Manage Ralstonia Disease(2005)

- Cassava variety KM140(2010)

Centres
Website links
Vietnamese calendar
Library
Visitors summary
 Curently online :  6
 Total visitors :  7515829

Successful production of genome-edited rats by the rGONAD method
Friday, 2018/08/24 | 07:46:04

Tomoe Kobayashi, Masumi Namba, Takayuki Koyano, Masaki Fukushima, Masahiro Sato, Masato Ohtsuka and Makoto Matsuyama

BMC Biotechnology  2018 18:19

https://doi.org/10.1186/s12896-018-0430-5

Background

Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “improved GONAD” (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats.

Results

Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in.

Conclusions

We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals.

 

See https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-018-0430-5

Figure 1: Determination of optimal electroporation efficiency for rGONAD. a Scheme of experimental procedures for evaluation of electroporation efficiency using GONAD method. b SZX7 stereomicroscope and Super Electroporator NEPA21. c Tetramethylrhodamine-labelled dextran is instilled into the oviduct lumen using a micropipette inserted through the oviductal wall near the infundibulum. d After the injection, the oviductal regions were covered with a piece of wet paper, and then, electroporation is performed using tweezer-type electrodes. e, f Scheme (e) or diagram (f) of electroporation conditions delivered of by an electroporator. This electroporation parameters were; Poring pulse; 40 V, 5 msec pulse, 50 msec pulse interval, number of pulse 3 times, 10% decay (± pulse orientation) and Transfer pulse; 10 V, 50 msec pulse, 50 msec pulse, number of pulse 6 times, 40% decay (± pulse orientation). g, h Fluorescence analysis of Tetramethylrhodamine-labelled dextran using GONAD method. i-k Graph shows analysis of the percentage of electroporation efficiency in WKY (i), DA (j), and DA x WKY (k). Ova, ovary; Ovi, Oviduct; Ute, uterus. Scale bars: 50 μm (g, h)

Back      Print      View: 448

[ Other News ]___________________________________________________
  • Auxin depletion from leaf primordia contributes to organ patterning
  • Phytochrome controls alternative splicing to mediate light responses in Arabidopsis
  • Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year
  • Identification and evaluation of quantitative trait loci underlying resistance to multiple HG types of soybean cyst nematode in soybean PI 437655
  • Factor analytic mixed models for the provision of grower information from national crop variety testing programs
  • Exploring the areas of applicability of whole genome prediction methods for Asian rice (Oryza sativa L.)
  • Investigation of terpene diversification across multiple sequenced plant genomes
  • Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism
  • Effect of the ahas Transgene on Biological Nitrogen Fixation and Yield of Soybean
  • Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity
  • A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome
  • High-Resolution Linkage Map and Chromosome-Scale Genome Assembly for Cassava (Manihot esculenta Crantz) from 10 Populations
  • Analysis of the Transcriptome of Banana Fruit during Ripening
  • Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1
  • Salt Tolerant Gene in Soybean Identified
  • Climate change decouples oceanic primary and export productivity and organic carbon burial
  • Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus
  • 5-Hydroxymethylcytosine Is Not Present in Appreciable Quantities in Arabidopsis DNA
  • Fine mapping of the qLOP2 and qPSR2 1 loci associated with chilling stress tolerance of wild rice seedlings
  • Natural diversity in daily rhythms of gene expression contributes to phenotypic variation

 

Designed & Powered by WEBSO CO.,LTD