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GRK5-Knockout Mice Generated by TALEN-Mediated Gene Targeting
Nanjidsuren, Tsevelmaa,Park, Chae-Won,Sim, Bo-Woong,Kim, Sun-Uk,Chang, Kyu-Tae,Kang, Myung-Hwa,Min, Kwan-Sik Taylor Francis 2016 Animal biotechnology Vol.27 No.4
<P>Transcription activator-like effector nucleases (TALENs) are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. We investigated the generation of mice with genetic knockout (KO) of the G protein-coupled receptor kinase (GRK) 5 gene by microinjection of TALEN mRNA. TALEN vectors were designed to target exons 1, 3, and 5 of the mouse GRK5 gene. Flow cytometry showed that the activity of the TALEN mRNAs targeted to exons 1, 3, and 5 was 8.7%, 9.7%, and 12.7%, respectively. The TALEN mRNA for exon 5 was injected into the cytoplasm of 180 one-cell embryos. Of the 53 newborns, three (5.6%) were mutant founders (F-0) with mutations. Two clones from F(0)28 showed a 45-bp deletion and F(0)39 showed the same biallelic non-frame-shifting 3-bp deletions. Three clones from F(0)41 were shown to possess a combination of frame-shifting 2-bp deletions. All of the mutations were transmitted through the germline but not to all progenies (37.5%, 37.5%, and 57.1% for the F(0)28, F(0)39, and F(0)41 lines, respectively). The homozygote GRK5-KO mice for 28 and 41 lines created on F3 progenies and the homozygous genotype was confirmed by PCR, T7E1 assay and sequencing.</P>
Analysis of Promoter Characterization of Macaque Monkey 20α-HSD Gene
Tsevelmaa Nanjidsuren,Sung-Jo Yun,Min-Su Kim,Chae-Won Park,Young-Sil Kang,Jong-Taek Yoon,Su-Yeon Hwang,Kwan-Sik Min 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s
20ɑ-hydroxysteroid dehydrogenase (20ɑ-HSD) enzyme converts progesterone into biological inactive steroid, thus playing a key role in the termination of pregnancy or estrus cycle and allowing parturition and ovulation to occur in most mammalian animals. However, function and regulation of this enzyme has not known well in primate reproductive physiology. We previously demonstrated the expression level and localization of the 20α-HSD in the reproductive tissues of macaque monkeys of pre-ovulation and pre-parturition period. Also, we amplified about 2005 bp 5'-flanking region from placenta genomic DNA and examined methylation pattern and promoter activity. In present study, we focus on the analysis of molecular characterization of the promoter region by using reporter assay systems. We constructed of deleted mutants (- 890 bp; HSF-2), (- 513 bp; XFD), (- 276 bp; Ap-1) and (- 72 bp; Sp-1) and each mutants were cloned into pGL3-basic vector. These deletion mutants were transfected into CHO cells and co-transfected with Sp-1 or Ap-1 transcription factor plasmids. Compared to - 890 bp and 513 bp promoter fragments alone, transcription activity increased when these constructs were co-transfected with Sp-1 and Ap-1 factor. However, for the absence Ap-1 factor binding site in 276 bp fragment activity dramatically decreased in both transfections. Next, we constructed of 306 bp fragment which is including of Ap-1 binding site and nucleotides converted mutants of the Ap-1 factor binding site. In this result, 306 bp fragment's transcription activity was high as wild type. However, the mutant activity which converted Ap-1 site’s all nucleotide was significantly decreased. These findings are confirmed by gel-shift assay examining Ap-1 binding site on the 20 α-HSD gene upstream region and expression of Ap-1 factor was determined by RT-PCR and Western blot in pre-parturition period placenta and CHO-K1 cell line. Our results indicate that Ap-1 site (- 281 → - 274) (5'-TGTCTCAT-3') plays a crucial role for monkey 20 α- HSD gene transcription.
TALEN-Mediated Gene Editing Method for GRK5-KO Mice
Tsevelmaa Nanjidsuren,Bo-Woong Sim,Min-Su Kim,Chae-Won Park,Eun-Bi Seo,Sun-Ok Kim,Kyu-Tae Chang,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2013 Reproductive & Developmental Biology(Supplement) Vol.37 No.2s
G protein-coupled receptor kinase 5 (GRK5) is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs). In recently, GRK5 deficiency has been linked to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may accelerate to AD pathogenesis remains elusive. The GRK5 mRNA is expressed widely in brain and peripheral tissues, with highest expression evident heart, lung, and placenta. In cellular model systems, GRK5 can phosphorylate several neuronal GPCRs including ß2-adrenergic, M2-muscarinic, secretin, angiotensin AT1, and thyroid stimulating hormone receptors. Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonucleases with the modular DNA-binding domain of TALEs and highly effective in inducing mutations at specific genome loci. TALEN-mediated mutagenesis in zygotes is a potential alternative to conventional gene targeting in mice. In the presented study, we report the generation of mice with genetic knockout of the GRK gene using TALENs. We designed TALEN vectors for exon 1, 3 and 5 of mouse GRK5 gene and tested their ability to alter the each surrogate vector in 293T cells. We prepared of mRNAs for the linearized TALEN using the mMessage mMachine T7 Ultra kit. mRNAs (4ng/μl) was injected into cytoplasm of 180 one-cell embryos. After incubation for 24 hours, the selected two-cell embryos transferred into the oviduct of seven pseudopregnant C57BL/6 mice. We confirmed the genotype of Fo mice by sequencing and T7E1 assay. We found 6 mutant mice lines (11%) from 53 newborns. We also mated 3 Fo GRK5 mutant lines with wild type mice and confirmed the genotype of the F1 progenies. All the mutations observed in Fo mice were transmitted through the germline but not all progenies (8/3, 13/4, 7/4). Taken together, TALEN-mediated mutagenesis might accelerate the creation of genetically engineered mouse models and elucidate the mechanism of AD pathogenesis using GRK5 knock out mice.
Comparative Analysis of the Aranesp, tg-EPO and Dimeric EPO Analogues In Vivo
Tsevelmaa Nanjidsuren,Purevjargal Naidansuren,Sung-Jo Yun,Jong-Ju Park,Won-Gun Noh,Chae-Won Park,Young-Sil Kang,Jong-Taek Yoon,Sang-Rae Lee,Kyu-Tae Chang,Kwan Sik-Min 한국동물생명공학회(구 한국동물번식학회) 2009 발생공학 국제심포지엄 및 학술대회 Vol.2009 No.1
Regulation of Monkey 20α-Hydroxysteroid Dehydrogenase (HSD) Promoter by PGF2a and Foskolin
Eun-Bi Seo,Tsevelmaa Nanjidsuren,Chae-Won Park,Min-Su Kim,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2013 Reproductive & Developmental Biology(Supplement) Vol.37 No.2s
The monkey 20ɑ-hydroxysteroid dehydrogenase (20ɑ-HSD) is an enzyme responsible for the catabolism in converting progesterone into biological inactive 20a-hydroxyprogesterone, thus playing a key role during the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. We have previously shown that 20ɑ-HSD in pre-ovulation and pre-parturition was highly detected in ovarian and placental tissues and localized mainly in the syncytiotrophoblast of the placenta. In this study, we focus on the analysis of molecular characterization of the monkey 20ɑ-HSD promoter region by using reporter assay systems in a Chinese hamster ovary (CHO) K1 cells. A reporter assay, using constructs (—890-Luc, —513-Luc, —306-Luc, —273-Luc and —70-Luc) of various lengths of the 5'-flanking region, revealed that the region (Ap-1) between —281 and —274 bp was essential for transcriptional activity. The absence of Ap-1 site in -273-Luc dramatically decreased to the control levels. Thus, Ap-1 site (—281→—274) (5'-TGTCTCAT-3') plays a crucial role for monkey 20α-HSD gene transcription. Next, we analyzed the monkey 20α-HSD promoter activity with treatment of prolactin, PGF2α and Forskolin in CHO-K1 cells. The 2005bp monkey promoter-pGL3 was transfected to CHO cells and added various dose-dependent treatments after incubation 24 hours. For PGF2α 0.5, 1, 5, 10 and 50 μM added and incubated during 6 hours. For Forskolin 0.1, 1, 10, 50 and 100 μM added. And finally, the luciferase activity was measured after 24 hours. Also, human prolactin (3μg/ml) was used as inhibitor. In result, PGF2a was response to a dose-dependent activation. On the other hand Forskolin was detected dose-dependent inhibition in the 2005bp 20α-HSD promoterpGL3 construct. However, prolactin was not shown any significant different. Taken together, our results indicate that monkey 20α-HSD promoter activity inhibit by forskolin. Also, PGF2a remarkedly stimulates the activity of monkey 20α-HSD promoter. We demonstrated that monkey 20a-HSD promoter regulate by AP-transcription factor, PGF2a, and Foskolin.
Production of Cloned Mice by Aggregation of Tetraploid Em
Bo-Woong Sim,Tsevelmaa Nanjidsuren,Chae-Won Park,Eun-Bi Seo,Min-Su Kim,Kye-Tae Chang,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2013 Reproductive & Developmental Biology(Supplement) Vol.37 No.2s
This study was investigated to optimize the efficiency of cloning and to produce the clone mice. Somatic cell nucleus transfer (SCNT) embryos were produced by injecting cumulus cell nucleus into enucleated B6D2F matured oocyte. Mouse chimeras can be also successfully produced with tetraploid host embryos. The fusion of mouse 2- cell embryos can be produced tetraploid embryos. Thus, we utilized the tetraploid embryos to increase the clone efficiency by aggregation with SCNT embryos of adult cumulus cells. SCNT embryos were found to be optimized for 6 hours activation with strontium (SrCl2). The cytochalasin B (CB) concentration (5 ug/ml) in the enucleation was evaluated in the efficiency of implantation sites and fetus offspring. Trichostatin A (TSA), histonedeacetylase inhibitor (HDACi), is recommended to production of clone mice, continuously exposed in 5~50 nM for 10 hours. And we also improved the SCNT implantation rate/ live offspring by co-transfer with parthenogenic embryo in uterus of the other site. Next, the aggregated SCNT were constructed by aggregation of SCNT embryo with tetraploid embryos to reduce epigenetic error in placenta. The pregnancy and implantation rates of aggregated SCNT were significantly higher than SCNT alone. The full term developmental rate in aggregated embryo was slightly higher than that of SCNT (3.57 vs 1.16). The placental weight of SCNT clone was significantly higher than that of in vitro fertilization as shown in previously reported. However, the placenta weight was almost reduced to IVF group in the aggregated SCNT. It was shown to be typical hyperplastic histology of mouse clones. But the aggregated SCNT method was useful to significantly reduce the placental weight known as general problems in cloned mice.