• Genomics & Informatics
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• v.10 no.1
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• pp.16-22
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• 2012

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of multiple fluid-filled cysts that expand over time and destroy renal architecture. The proteins encoded by the $PKD1$ and $PKD2$ genes, mutations in which account for nearly all cases of ADPKD, may help guard against cystogenesis. Previously developed mouse models of $PKD1$ and $PKD2$ demonstrated an embryonic lethal phenotype and massive cyst formation in the kidney, indicating that $PKD1$ and $PKD2$ probably play important roles during normal renal tubular development. However, their precise role in development and the cellular mechanisms of cyst formation induced by $PKD1$ and $PKD2$ mutations are not fully understood. To address this question, we presently created $Pkd2$ knockout and $PKD2$ transgenic mouse embryo fibroblasts. We used a mouse oligonucleotide microarray to identify messenger RNAs whose expression was altered by the overexpression of the $PKD2$ or knockout of the $Pkd2$. The majority of identified mutations was involved in critical biological processes, such as metabolism, transcription, cell adhesion, cell cycle, and signal transduction. Herein, we confirmed differential expressions of several genes including aquaporin-1, according to different $PKD2$ expression levels in ADPKD mouse models, through microarray analysis. These data may be helpful in $PKD2$-related mechanisms of ADPKD pathogenesis.

• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.99-105
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• 2014

In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhibition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

• IMMUNE NETWORK
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• v.2 no.4
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• pp.233-241
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• 2002

Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain ${\lambda}$ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.453-458
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• 1997

It is known that the incorporation of genes into transgenic mice is generally stable and is p passed on to succeeding generations in a Mendelian fashion. In this report, transgenic mice were set as a model to evaluate whether the transgenes are transmitted in a Mendelian principle in a successive generations and how they are tran s smitted into their offspring. A 3.0 kb linear DNA fragment, containing the MMTV LTR, bovine aSI casein cDNA and SV 40 splicing and polyadenylation site; was microinjected into fertilized mouse embryos. The tail DNAs of the resulting pups were subjected to dot and Southern hybridizations to screen transgenic founders. The DNAs of their offspring were anlyzed by PCR to confirm the transmission of the transgene from F0. Out of 72 live pups four pups (5.6%), 3 males and 1 female, were positive for the transgene. The rates of transmission from F0 into F1 were 33.3, 7.7, 0, and 62.5%. Those from F1 into F2 were 63.6, 5.9, and 68.8% and those from F2 into F3 were 85.7, and 88.2%. In this report, the transmission pattern of transgenes in transgenic mice into their offspring was demonstrated. It either follows or does not follow in a Mendelian fashion. Deletion or loss of the transgenes from F0 in some lines became apparant to the succeeding generations.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.95-104
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• 2002

This study was performed during the four seasons for the production of transgenic pigs containing the human erythropoietin(hEPO) transgene. Purebred Landrace gilts and sows approximately 8∼15 months of age (n=42) were used fur the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Synchronization and superovulation procedures were used that cyclic gilts were synchronized with 20mg altrenogest (ALT) per day for 9days after PG600 administration followed by superovulation with 1500IU pregnant mares serum gonadotropin (PMSG) and 500IU human chorionic gonadotrophin (hCG). Preparation of recombinant gene for microinjection is mice whey acidic protein promoter (mWAP) linked to human erythropoietin (hEPO) gene. After hormone treatment, 650 embryos were collected from 23 donors and 83.1% (540/650) embryos were in 1-cell stage which can be visualized the pronuclei for DNA microinjection. A total of 543 DNA microinjected embryos fiom donors were transferred to 19 synchronized recipients, seven of them maintained pregnancy and delivered 47 piglets. One of the 47 offsprings were determined to have transgene by PCR analysis. The overall rate of transgenic production was 2.13% (tansgenic/offspring). This study provides the success and useful information regarding production of transgenic pig for bioreactor research.

• Proceedings of the Korean Society of Toxicology Conference
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• 1998.10a
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• pp.36-36
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• 1998

Tumorigenicity of benzo(a)pyrene (BP) and benzo(a)pyrene diol epoxides ((+)BPDE-1, (-)BPDE-1) was investigated in transgenic TG-AC mice carrying v-Ha-ras oncogene fused to the promoter of the mouse embryonic a-like, z-globin gene. Animals were topically treated twice per week for 25weeks with BPDE (10$\mu$g/mouse) and BP (10, 20, 40$\mu$g/mouse). In addition, animals were treated with BPDE or BP (initiated) followed by TPA (2$\times$2.5$\mu$g/week, for 4 weeks) for promotion study. In the continuous treatment of BPDE or BP, animals treated with 40$\mu$g BP showed $100\%$ tumor response after 20 weeks, $40\%$ of mice for 20$\mu$g BP, and $20\%$ for (+)BPDE-1, but (-)BPDE-1 and 10$\mu$g BP did not show any tumor response. After 25 weeks, most tumors turned out to be carcinomas in animals treated with 40$\mu$g BP. In BPDE or BP/TPA Initiation-promotion study, papilloma response occurred earlier (6 weeks after TPA treatment) than in continuously treated animals with BPDE or BP. RT-PCR assay for transgene expression showed that BP or BPOE was not transgene dependent in its tumorigenicity, but TPA was. Several Cytokine genes(TGF-a, TNF-a) and c-myc gene expressions were monitored in skin tissues during BP carcinogenesis. In early stage of BP treatment, the gene expressions were elevated(c-myc,TGF-a) or unchanged(TNF-a) compared to control, but the levels were gradually decreased during both middle and late stages of cacinogenesis, Gene expression levels of skin papillomas in acetone initiated-TPA promoted animals were close to those of middle stage or between middle and late stages. i-NOS was also highly expressed in carcinoma and papilloma, These data suggest that transgene expressions of TG-AC mice were not dependent on BP carcinogenesis and that TG-AC mice were more sensitive to TPA regardless of types of initiators. In addition, genes(TGF-a, c-myc, TNF-a, i-NOS) were modulated in the skin during BP cacinogenesis or TPA promotion.

• Journal of Life Science
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• v.32 no.8
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• pp.611-621
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• 2022

Dimethyl sulfoxide (DMSO) is commonly used as control or vehicle solvent in preclinical research of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) due to its ability to dissolve lipophilic compounds and cross the blood brain barrier. However, the biochemical effects of DMSO on the outcomes of preclinical research are often overlooked. In the present study, we investigated whether the long-term oral administration of 5% DMSO affects the neurological, functional, and histological disease phenotype of the copper/zinc superoxide dismutase glycine 93 to alanine mutation (SOD1-G93A) mouse model of amyotrophic lateral sclerosis. SOD1-G93A transgenic mice showed shortened survival time and reduced motor function. We found that administration with DMSO led to increased mean survival time, reduced neurological scores, and improved motor performance tested using the rotarod and grip strength tests. On the other hand, DMSO treatment did not attenuate motor neuron loss in the spinal cord and denervation of neuromuscular junctions in the skeletal muscle. These results suggest that DMSO administration could improve the quality of life of the SOD1-G93A mouse model of ALS without affecting motor neuron denervation. In conclusion, the use of DMSO as control or vehicle solvent in preclinical research may affect the behavioral outcomes in the SOD1-G93A mouse model. The effect of the vehicle should be thoroughly considered when interpreting therapeutic efficacy of candidate drugs in preclinical research.

• BMB Reports
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• v.36 no.1
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• pp.35-42
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• 2003

Heterocyclic amines (HCAs) are potent mutagens generated during the cooking of meat and fish, and several of these compounds produce tumors in conventional experimental animals. During the past 5 years or so, HCAs have been tested in a number of novel in vivo murine models, including the following: lacZ, lacI, cII, c-myc/lacZ, rpsL, and $gpt{\Delta}$ transgenics, $XPA^{-/-}$, $XPC^{-/-}$, $Msh2^{+/-}$, $Msh2^{-/-}$ and $p53^{+/-}$ knock-outs, Apc mutant mice ($Apc^{{\Delta}716}$, $Apc^{1638N}$, $Apc^{min}$), and $A33^{{\Delta}N{\beta}-cat}$ knock-in mice. Several of these models have provided insights into the mutation spectra induced in vivo by HCAs in target and non-target organs for tumorigenesis, as well as demonstrating enhanced susceptibility to HCA-induced tumors and preneoplastic lesions. This review describes several of the more recent reports in which novel animal models were used to examine HCA-induced mutagenesis and carcinogenesis in vivo, including a number of studies which assessed the inhibitory activities of chemopreventive agents such as 1,2-dithiole-3-thione, conjugated linoleic acids, tea, curcumin, chlorophyllin-chitosan, and sulindac.

• The korean journal of orthodontics
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• v.33 no.6 s.101
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• pp.485-497
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• 2003

The form and function of the craniofacial structure critically depend on genetic information. With recent advances in the molecular technology, genes that are important for normal growth and morphogenesis of the craniofacial skeleton are being rapidly uncovered, shaping up modem craniofacial biology. One of them is fibroblast growth factor receptor 2 (FGFR2). Specific point mutations in the. FGFR2 gene have been linked to Apert syndrome, which is characterized by premature closure of cranial sutures and craniofacial anomalies as well as limb deformities. To study pathogenic mechanisms underlying craniosynostosis phenotype of Apert syndrome, we used a transgenic approach; an FGFR2 minigene construct containing an Apert mutation (a point mutation that substitute proline at the position 253 to arginine; P253R) was introduced into fertilized mouse germ cells by DNA microinjection. The injected cells were then allowed to develop into transgenic mice. We used a bone-specific promoter (a DNA fragment from the type I collagen gene) to confine the expression of mutant FGFR2 gene to the bone tissue, and asked whether expression of mutant FGFR2 in bone is sufficient to cause the craniosynostosis phenotype in mice. Initial characterization of these mice shows prematurely closed cranial sutures with facial deformities expected from Apert patients. We also demonstrate that the transgene produces mutant FGFR2 protein with increased functional activities. Having this useful mouse model, we now can ask questions regarding the role of FGFR2 in normal and abnormal development of cranial bones and sutures.

• BMB Reports
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• v.45 no.3
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• pp.153-158
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• 2012

Geft is a guanine nucleotide exchange factor, which can specifically activate Rho family of small GTPase by catalyzing the exchange of bound GDP for GTP. Geft is highly expressed in the excitable tissue as heart and skeletal muscle and plays important roles in many cellular processes, such as cell proliferation, migration, and cell fate decision. However, the in vivo role of Geft remains unknown. Here, we generated a Geft conditional knockout mouse by flanking exons 5-17 of Geft with loxP sites. Cre-mediated deletion of the Geft gene in heart using Mef2c-Cre transgenic mice resulted in a dramatic decrease of Geft expression. Geft knockout mice develop normally and exhibit no discernable phenotype, suggesting Geft is dispensable for the development of the second heart field in mouse. The Geft conditional knockout mouse will be a valuable genetic tool for uncovering the in vivo roles of Geft during development and in adult homeostasis.