• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.109-117
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• 2020

Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TAT-Cre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TAT-Cre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TAT-Cre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.886-892
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• 2017

Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.147-159
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• 2018

Genetically engineered mouse models are commonly preferred for studying the human disease due to genetic and pathophysiological similarities between mice and humans. In particular, Cre-loxP system is widely used as an integral experimental tool for generating the conditional. This system has enabled researchers to investigate genes of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner. A various tissue-specific Cre-driver mouse lines have been generated to date, and new Cre lines are still being developed. This review provides a brief overview of Cre-loxP system and a few commonly used promoters for expression of tissue-specific Cre recombinase. Also, we finally introduce some available links to the Web sites that provides detailed information about Cre mouse lines including their characterization.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.826-830
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• 2018

A Cre/loxP-${\delta}$-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae. To allow repeated integrations, the reusable Candida glabrata MARKER (CgMARKER) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and ${\beta}$-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

• BMB Reports
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• v.34 no.4
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• pp.322-328
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• 2001

Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage P1 and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive $P_{hcmv^*-1}$ promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy.

• Journal of Life Science
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• v.17 no.4 s.84
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• pp.587-592
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• 2007

Manipulation of the mouse genome by activating or inactivating the gene has contributed to the understanding of the function of the gene in the subset of cells during embryonic development or postnatal period of life. Most of all, gene targeting, which largely depends on the availability of mouse embryonic stem (ES) cells, is the milestone of development of animal models for human disease. Recombinase-mediated genome modification (Cre-LoxP and Flp-Frt etc) and the ligand-dependent regulation system, more accurate and elaborate manipulation tools, have been successfully developed and applied to dissect the mechanisms governing complex biological processes and to understand the role of protein in temporal-and spatial aspects of development. As technologies concerning refined manipulation of mouse genome are developed, they are expected to open new opportunities to better understand the diverse in vivo functions of genes.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.3
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• pp.170-176
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• 2023

Objective: Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7^f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. Methods: All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7^f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. Results: SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. Conclusion: Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.745-753
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• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• Journal of Korean Dental Science
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• v.9 no.1
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• pp.9-18
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• 2016

Purpose: Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. Deletion of the Wntless (Wls) gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation. However, it remains unclear if autonomous Wnt ligands are necessary for differentiation of dental pulp cells into odontoblast-like cells to induce reparative dentinogenesis, one of well-known feature of pulp repair to form tertiary dentin. Materials and Methods: To analyze the autonomous role of Wls for differentiation of dental pulp cells into odontoblast-like cells, we used primary dental pulp cells from unerupted molars of Wls-floxed allele mouse after infection with adenovirus for Cre recombinase expression to knockout the floxed Wls gene or control GFP expression. The differentiation of dental pulp cells into odontoblast-like cells was analyzed by quantitative real-time polymerase chain reaction. Result: Proliferation rate was significantly decreased in dental pulp cells with Cre expression for Wls knockout. The expression levels of Osterix (Osx), runt-related transcription factor 2 (Runx2), and nuclear factor I-C (Nfic) were all significantly decreased by 0.3-fold, 0.2-fold, and 0.3-fold respectively in dental pulp cells with Wls knockout. In addition, the expression levels of Bsp, Col1a1, Opn, and Alpl were significantly decreased by 0.7-fold, 0.3-fold, 0.8-fold, and 0.6-fold respectively in dental pulp cells with Wls knockout. Conclusion: Wnt ligands produced autonomously are necessary for proper proliferation and odontoblastic differentiation of mouse dental pulp cells toward further tertiary dentinogenesis.

• BMB Reports
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• v.49 no.11
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• pp.585-586
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• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.