Preparation of PrP-specific Polyclonal Antibody Immunization of -knockout Mice with Recombinant Human PrP Protein / 生物医学与环境科学（英文）

Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Spatio-temporal expression of dentin sialophosphoprotein and collagen Ⅰ during molar tooth germ development in vps4b knockout mouse / 华西口腔医学杂志

OBJECTIVE@#To verify the effect of the mutant gene vps4b on the expression of tooth development-related proteins, dentin sialophosphoprotein (DSPP) and collagenⅠ (COL-Ⅰ).@*METHODS@#Paraffin tissue sections of the first molar tooth germ were obtained from the heads of fetal mice at the embryonic stages of 13.5, 14.5, and 16.5 days and from the mandibles of larvae aged 2.5 and 7 days after birth. The immunohistochemical method was used to detect the expression and location of DSPP and COL-Ⅰ in wild-type mouse and vps4b knockout mouse.@*RESULTS@#DSPP and COL-Ⅰ were not found in the bud and cap stages of wild-type mouse molar germ. In the bell stage, DSPP was positively expressed in the inner enamel epithelium and dental papilla, whereas COL-Ⅰ was strongly expressed in the dental papilla and dental follicle. During the secretory and mineralized periods, DSPP and COL-Ⅰ were intensely observed in ameloblasts, odontoblasts, and dental follicles, but COL-Ⅰ was also expressed in the dental papilla. After vps4b gene knockout, DSPP was not expressed in the dental papilla of the bell stage and in the dental papilla and dental follicle of the secretory phase. The expression position of COL-Ⅰ in the bell and mineralization phase was consistent with that in the wild-type mice. Moreover, the expression of COL-Ⅰ in the dental papilla changed in the secretory stage.@*CONCLUSIONS@#Gene vps4b plays a significant role in the development of tooth germ. The expression of DSPP and COL-Ⅰ may be controlled by gene vps4b and regulates the development of tooth dentin and cementum together with vps4b.

Disruption of the Tff1 gene in mice using CRISPR/Cas9 promotes body weight reduction and gastric tumorigenesis / 한국실험동물학회지

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. TFF1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated TFF1-knockout (KO) mice, without a neomycin resistant (NeoR) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our TFF1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established TFF1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that TFF1 expression influences gender differences.

Collagen-Induced Arthritis Analysis in Rhbdf2 Knockout Mouse

Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha (TNF-α) converting enzyme, which is the molecule responsible for the release of TNF-α. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of TNF-α release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative TNF-α related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to TNF-α.

Extracting Extra-Telomeric Phenotypes from Telomerase Mouse Models

Telomerase reverse transcriptase (TERT) is the protein component of telomerase and combined with an RNA molecule, telomerase RNA component, forms the telomerase enzyme responsible for telomere elongation. Telomerase is essential for maintaining telomere length from replicative attrition and thus contributes to the preservation of genome integrity. Although diverse mouse models have been developed and studied to prove the physiological roles of telomerase as a telomere-elongating enzyme, recent studies have revealed non-canonical TERT activities beyond telomeres. To gain insights into the physiological impact of extra-telomeric roles, this review revisits the strategies and phenotypes of telomerase mouse models in terms of the extra-telomeric functions of telomerase.

Studies on In Vivo Function of Peroxiredoxins in Knockout Mice / 한양의대학술지

Peroxiredoxins (Prxs) are a family of antioxidant proteins that reduce peroxide levels by using reducing agents such as thioredoxin. These proteins were characterized to have a number of cellular functions, including cell proliferation and differentiation and protection of specific proteins from oxidative damage. Thus, it is important to clarify the physiological role of Prxs by generating mouse models deficient in each Prx to better understand the in vivo function of Prxs. We have generated and characterized mice deficient in Prx I and II that are abundantly expressed in almost all types of cells. The Prx II-/- mice were healthy in appearance and fertile, however showed several pathophysiological disorders. Using the mice, we found that Prx II is an essential antioxidant enzyme that prevents oxidative stress in erythropoiesis, protects against endotoxin-induced lethal shock, regulates platelet-derived growth factor signaling and angiogenesis, inhibits cellular senescence, preserves cognitive function against age-linked hippocampal oxidative damage and exacerbates tumorigenesis in a liver cancer mouse model. The Prx I-/- mice were also healthy in appearance and fertile like Prx II-/- mice. With the mice, we found that Prx I suppresses K-ras-driven lung tumorigenesis by opposing the redox-sensitive extracellular-signal-regulated kinase/cyclin D1 pathway and plays concerted action with sulfiredoxin in preventing against alcohol-induced oxidative injury in the mouse liver. The results obtained suggest that Prx I and II are essential antioxidant enzymes for maintaining redox homeostasis in mice.

Difference in Resistance to Streptococcus pneumoniae Infection in Mice / 한국실험동물학회지

Streptococcus pneumoniae is a major pathogen that causes various diseases, including pneumonia and sepsis, as millions of people suffer from S. pneumoniae infection worldwide. To better understand the immune and inflammatory responses to S. pneumoniae, we produced murine models. To investigate the differences between intranasal and intratracheal infection, BALB/c mice were infected with S. pneumoniae D39 intranasally or intratracheally. Mice showed no significant differences in survival rates, body weight changes, and bacterial loads. To investigate resistance and susceptibility among mouse strains, BALB/c, C57BL/6J, tumor necrosis factor-alpha (TNF-alpha) knockout, and interleukin-10 (IL-10) knockout mice were infected with S. pneumoniae D39 via intranasal or intravenous routes. In this study, BALB/c and C57BL/6J mice were resistant, IL-10 knockout mice were intermediate, and TNF-alpha knokout mice were susceptible to S. pneumoniae infection. These data show that intranasal and intratracheal infection induced similar results after S. pneumoniae infection, and the genetic background of mice must be considered when studying S. pneumoniae infection in vivo.

Expression, purification Orai2 protein and preparation, application it's polyclonal antibody / 中国免疫学杂志

Objective:To prepare GST-Orai2 fusion protein and to prepare polyclonal antibody against Orai2 by immunizing rabbits.To further investigate the function of Orai2,a transmembrane protein,this antibody was used to identify the Orai2 conditional gene knockout mice.Methods:ORF of Orai2 was amplified by PCR and subcloned into pGEX-6p-1 vector.After transforming BL21 (DE3) competent cells,we succeeded in inducing the expression of GST-Orai2 fusion protein using IPTG.Then the GST-Orai2 protein was purified by immobilized Glutathione affinity chromatography and identified by SDS-PAGE.A New England rabbit was immunized with the prepared fusion protein in Freund's adjuvant to prepare specific antibody.Finally,the prepared antibody was identified by Western blot by checking its titer and specificity.Furthermore,we took use of the prepared Orai2 antibody to identify Orai2 conditional gene knockout mice,with comparing to the wildtype ones in the same cage.Results:The purity of purified GST-Orai2 reached to 90% and the concentration was 0.35 mg/ml by BCA kit.We could detect Orai2 protein even in dilution of 1:10,000.Also,the prepared polyclonal antibody agianst Orai2 could detect both overexpressed and endogenous Orai2 protein in mouse-brain,without crossing reaction with Orai1.As well,we found that the Orai2 protein expression was of obvious reduction in Orai2 conditional gene knockout mice,compared with the wildtype ones in the same cage.Conclusion:We successfully obtain the purified GST-Orai2 fusion protein and prepare specific and highly sensitive polyclonal antibody against Orai2.The antibody can be used to detect overexpressed and endogenous Orai2 protein inmouse-brain specifically,and to identify Orai2 conditional gene knockout mice,without any crossing reaction with Orai1.Our work contributes a lot to the future investigation of functions of Orai2.

Morphologic Change of the Vestibular Organ in the Na+-K+-2Cl- Cotransporter Deficiency Mouse

BACKGROUND AND OBJECTIVES: The Na+-K+-2Cl- cotransporter-1 (NKCC1) is a member of the cation-coupled chloride transporter that participates in salt transport and cell volume regulation in diverse tissues. NKCC1 deficient mice exhibit deafness, and have structural alterations in the cochlea. In addition to hearing loss, NKCC1-deficient mice show a shaker-waltzer behavior, which suggests a vestibular system defect. This study investigated the morphology of the vestibular system of NKCC1-deficient mice. In addition, this study evaluated whether NKCC1 mRNA and its protein are expressed in human vestibular end organs. MATERIALS AND METHOD: NKCC1-deficient and wild type mice aged 4~5 weeks were sacrificed. Their heads were cut in the midsagittal plane, fixed and decalcified. For light microscopy, 5 m sections were cut, and stained with hematoxylin and eosin. Human vestibular end organs were harvested during acoustic tumor surgery via translabyrinthine approach. Some of these end organs were used for the total mRNA extraction and the remainder was used for immunostaining. RT-PCR was performed for NKCC1. RESULTS: The scala media of the cochlear of the NKCC1-deficient mice were collapsed but the bony labyrinth of the cochlea appeared unaffected. However, the semicircular canals (SCCs) were much smaller than those in the wild type. Furthermore, the SCCs were completely missing in some NKCC1-deficient mice. NKCC1 mRNA was expressed in both human macula and crista ampullaris and its protein was expressed mainly in the transitional and dark cell area of the human crista ampullaris. CONCLUSION: NKCC1 may be essential for maintaining the vestibular morphology and its function in mice and NKCC1 is well expressed in human vestibular end organs.

Morphologic Change of the Vestibular Organ in the Na+-K+-2Cl- Cotransporter Deficiency Mouse

BACKGROUND AND OBJECTIVES: The Na+-K+-2Cl- cotransporter-1 (NKCC1) is a member of the cation-coupled chloride transporter that participates in salt transport and cell volume regulation in diverse tissues. NKCC1 deficient mice exhibit deafness, and have structural alterations in the cochlea. In addition to hearing loss, NKCC1-deficient mice show a shaker-waltzer behavior, which suggests a vestibular system defect. This study investigated the morphology of the vestibular system of NKCC1-deficient mice. In addition, this study evaluated whether NKCC1 mRNA and its protein are expressed in human vestibular end organs. MATERIALS AND METHOD: NKCC1-deficient and wild type mice aged 4~5 weeks were sacrificed. Their heads were cut in the midsagittal plane, fixed and decalcified. For light microscopy, 5 m sections were cut, and stained with hematoxylin and eosin. Human vestibular end organs were harvested during acoustic tumor surgery via translabyrinthine approach. Some of these end organs were used for the total mRNA extraction and the remainder was used for immunostaining. RT-PCR was performed for NKCC1. RESULTS: The scala media of the cochlear of the NKCC1-deficient mice were collapsed but the bony labyrinth of the cochlea appeared unaffected. However, the semicircular canals (SCCs) were much smaller than those in the wild type. Furthermore, the SCCs were completely missing in some NKCC1-deficient mice. NKCC1 mRNA was expressed in both human macula and crista ampullaris and its protein was expressed mainly in the transitional and dark cell area of the human crista ampullaris. CONCLUSION: NKCC1 may be essential for maintaining the vestibular morphology and its function in mice and NKCC1 is well expressed in human vestibular end organs.

Adrenomedullin against insulin resistance / 中华内分泌代谢杂志

To reveal the involvement of oxidative stress in hypertension and insulin resistance. Angiotensin Ⅱ was given to adrenomedullin-knockout mice for 4 weeks. 4-Hydroxy-TEMPOL, a superoxide scavenger mimetic was also employed. The results suggested that adrenomedullin may exert a protective effect against insulin resistance via its intrinsic antioxidant effect.