Adult ICR Mice Research Articles

54 SUCCESS OF MOUSE CLONING FROM AN OUTBRED STRAIN BY TRICHOSTATIN A TREATMENT AFTER SOMATIC NUCLEAR TRANSFER

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, the extremely low success rates have plagued this technique for a decade. Further, in mice, the clonable strains have been limited mainly to the hybrid F1 strains such as B6D2F1. Recently, we have reported a new efficient cloning technique using trichostatin A (TSA) where reconstructed oocytes are activated by 5 mM strontium with 5 nM TSA for 6 h, followed by 3 h of culture in KSOM medium containing the same concentration of TSA. After the TSA treatment, cloned embryos were cultured in KSOM medium without TSA. This TSA treatment leads to a 2–5-fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. In this study, to further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4–5% success rates, which is comparable to 6–7% of B6D2F1. Thus, the TSA cloning technique now allows us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from the hybrid strains but also enables mouse cloning from normally unclonable strains. Further, our results provide insight into the mechanism underlying why only limited strains can be cloned using the current standard cloning technique.

Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia–reperfusion injury in mice

Induction of heat shock factor 1 (HSF1) is known to associate with cellular response to divergent pathophysiological stresses including whole body hyperthermia (WBH) and ischemia-reperfusion. However, a direct cause-effect relationship between HSF1 activation and cytoprotection induced by myocardial preconditioning has not been conclusively established, mainly due to the limitations of available experiment tools. In the present studies, we used a novel approach to block HSF1 with small interfering RNA (siRNA) technique in vivo. Male adult ICR mice were treated intraperitoneally with amine (vehicle) or siRNA specific to HSF1 (siRNA-HSF1). Three days later, WBH preconditioning protocol (rectal temperature 42 °C for 15 min) was applied to these mice under light anesthesia. WBH preconditioning resulted in 2.7-fold and 3.4-fold increase in cardiac HSF1 mRNA and protein expression respectively 2 hours after WBH, which was inhibited in the siRNA-treated mice. The silencing effect of siRNA on HSF1 was associated with complete loss of the infarct- limiting protection by WBH preconditioning after 48 hours. Pretreatment with siRNA-HSF1 had no effect on infarct size in the sham control animals as compared with the amine-treated group. DNA micro-array analysis revealed that siRNA-HSF1 caused a general inhibition on multiple members of HSP family, except Hsp32, Hsp47 and Hsp60. In addition, the silencing effect of siRNA on HSF1 and HSPs gene expression was transient and its inhibitory effect disappeared by 10 days after treatment. siRNA-HSF1 also impaired the thermotolerance of the heat shocked mice as indicated by higher mortality following WBH. For the first time, we have applied siRNA technique in the field of myocardial preconditioning to demonstrate HSF1 activation as an essential step in WBH preconditioning against cardiac ischemia-reperfusion injury.

Using siRNA Technique to Generate Transgenic Animals with Spatiotemporal and Conditional Gene Knockdown

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.

Importance of satellite cells in the strength recovery after eccentric contraction-induced muscle injury.

The purpose of this study was to determine if the elimination of satellite cell proliferation using gamma-irradiation would inhibit normal force recovery after eccentric contraction-induced muscle injury. Adult female ICR mice were implanted with a stimulating nerve cuff on the common peroneal nerve and assigned to one of four groups: 1) irradiation- and eccentric contraction-induced injury, 2) eccentric contraction-induced injury only, 3) irradiation only, and 4) no intervention. Anterior crural muscles were irradiated with a dose of 2,500 rad and injured with 150 in vivo maximal eccentric contractions. Maximal isometric torque was determined weekly through 35 days postinjury. Immediately after injury, maximal isometric torque was reduced by approximately 50% and had returned to normal by 28 days postinjury in the nonirradiated injured mice. However, torque production of irradiated injured animals did not recover fully and was 25% less than that of injured nonirradiated mice 35 days postinjury. These data suggest that satellite cell proliferation is required for approximately half of the force recovery after eccentric contraction-induced injury.

IMPORTANCE OF SATELLITE CELLS IN RECOVERY FROM ECCENTRIC CONTRACTION-INDUCED INJURY

Eccentric contractions cause injury to skeletal muscle evidenced by prolonged decrements in force. Satellite cells are activated in response to eccentric contraction-induced injury, but their importance in the recovery of force after injury is unknown. PURPOSE To determine if satellite cells are required for the recovery of force after eccentric contraction-induced injury. METHODS Adult female ICR mice were implanted with a stimulating nerve cuff on the common peroneal nerve and assigned to one of four groups: 1) irradiation and eccentric contraction-induced injury (IRR/INJ, n = 11), 2) eccentric contraction-induced injury only (N-IRR/INJ, n = 10), 3) irradiation only (IRR/N-INJ, n = 11), and 4) no intervention (N-IRR/N-INJ, n = 8). Anterior crural muscles were irradiated with a dose of 2500 rad and injured 48 hr later with 150 in vivo maximal eccentric contractions during electrical stimulation. Maximal isometric torque was determined at 0, 7, 14, 21, 28, and 35 days after the induction of injury. Group × time comparisons were made using a two-way ANOVA with repeated measures over time. RESULTS Maximal isometric torque production by N-IRR/INJ mice was not different from control animals by day 28 after injury induction (2.82 ± 0.37 vs. 2.91 ± 0.36 N mm). However, maximal isometric torque production of IRR/INJ animals did not recover and was less than N-IRR/INJ at 14 (19%), 21 (16%), 28 (21%), and 35 (25%) days after injury. CONCLUSION Satellite cells are required for normal force recovery following eccentric contraction-induced injury.

Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: Impact on gliogenesis in vivo

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2-5-day-old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express mu-, delta-, and/or kappa-receptor immunoreactivity in a cell type-specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5-bromo-2'-deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential mu-agonist) significantly decreased the number of BrdU-labeled GFAP(+) cells compared with controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100beta(+) cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP(-)/S100beta(-). When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP(-)/S100beta(-) cells is tonically suppressed by endogenous opioids. Assuming that S100beta and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors.

Developmental expression of meprin metalloprotease subunits in ICR and C3H/He mouse kidney and intestine in the embryo, postnatally and after weaning

Meprins are secreted and membrane-bound metalloendopeptidases highly expressed in kidney and intestinal epithelial cells. They are oligomeric glycoproteins composed of evolutionarily related α and/or β subunits. The present work revealed that the messages for both meprin subunits were expressed in intestine and kidney in ICR and C3H/He mouse embryos (as early as day 11), indicating developmental functions for both subunits. During the first 2 weeks after birth, the mRNA levels for both subunits increased in ICR mice, but between 10 days and 3 weeks (time of weaning) the α subunit level in the intestine fell markedly. In adult ICR mice, meprin β mRNA was consistently expressed in both kidney and intestine, whereas meprin α mRNA was highly expressed in kidney but only present at low levels in intestine. In C3H/He mice, the pattern of meprin α and β subunit mRNA expression was similar to that of ICR mice, except that meprin α was barely detectable in kidney after birth. The results of postnatal studies indicate that the meprin α subunit has a role in the intestine during suckling but is not essential after weaning, and that the β homooligomer is the major meprin form after weaning in both kidney and intestine.

Characterization of low dose streptozotocin-induced progressive diabetes in mice

Our previous study indicated that a single i.p. injection of 100 mg/kg streptozotocin (STZ) is able to induce slowly progressive diabetes mellitus in adult ICR mice. In the present study, the non-fasting serum insulin levels of the mice administered 100 mg/kg STZ were normal throughout the 24-week-observation after STZ injection. In the STZ-administered mice, the area of islets and the number of insulin-immunoreactive cells (β-cells) were normal at 1 week and then continued to decrease gradually as the time went on. In contrast, there was a relative increase in the number of glucagon-immunoreactive cells (α-cells) in these mice. In addition, in the STZ-administered mice, the degree of glucose tolerance continued to reduce from 2 weeks till 12 weeks when the experiment terminated. The rise in serum insulin levels stimulated by glucose in the STZ-administered mice began to subside from about 2 weeks and had completely ceased by 12 weeks. These results indicate that 100 mg/kg STZ-induced diabetic mouse model is non-insulin-dependent diabetes, which is characterized by impaired insulin response to glucose stimulation.

Microbiological quality and incidence of Staphylococcus aureus and Bacillus cereus in vegetarian food products

A total of 320 samples of vegetarian food purchased from local markets were examined in order to determine the microbiological quality of these products. Aerobic count, psychrotrophic count, anaerobic count, E. Coli, coliforms, S. aureus, B. cereus, spores of Bacillus as well as enterotoxins produced by B. cereus andS. aureus were also evaluated. The incidence of E. coli and coliforms in these vegetarian food products were 28·1% and 32·5%, respectively, while 18·1% and 3·4% of the samples were found to contain S. aureus and B. cereus, respectively. The vegetarian food products investigated in the present study were divided into five groups, based on their major components. The samples made from soybeans showed the highest detection rate (74·5%) when S. aureus, B. cereus, E. coli and coliforms were used as indicators. S. aureus was detected in all the five groups, with the highest detection rate in cereal and Konjac group (23·8%), while B. cereus was detected only in soybean, cereal and Konjac groups with the incidence of 5·1%, 9·5% and 2·4%, respectively. Among the 38 strains of S. aureus isolates, 17 strains were observed to produce enterotoxins of types A, B, C and D. Whereas, among the seven strains of B. cereus isolates, one produced diarrheal toxin while four others killed adult ICR mice. The present results also revealed that vacuum-packed vegetarian foods have the lowest incidence of micro-organisms tested in this study.

Molecular Cloning of a Leucine Zipper Motif-containing Novel cDNA Specifically Expressed in Adult Mouse Testis

A novel cDNA clone was isolated from a mouse testis cDNA library. This cDNA is 2,020 nucleotides in size and predicted to encode 527 amino acid residues with a molecular weight of 59.9 kDa. Protein sequence motif analysis revealed that the predicted protein contained one leucine zipper motif in its N-terminal portion and two basic domains in its C-terminal portion. Northern blot analysis of adult ICR mouse organs using the cDNA as probe demonstrated that an approximately 2.0-kb transcript was specifically expressed in testis. We therefore termed this novel cDNA tsec-2, testis-specifically expressed cDNAs-2. Results of Southern blot analysis suggested that the tsec-2 gene may exist as a single copy in the mouse genome.

Apoptosis plays an important role in experimental rabies virus infection.

Cultured rat prostatic adenocarcinoma (AT3) cells infected with the challenge virus standard (CVS) strain of fixed rabies virus showed characteristic morphologic features of apoptosis, evidence of oligonucleosomal DNA fragmentation, and expression of the Bax protein. CVS-infected Bcl-2-transfected AT3 cells did not demonstrate these features. Adult ICR mice inoculated intracerebrally with CVS showed morphologic changes of apoptosis, DNA fragmentation, and increased Bax expression in neurons, with changes most marked in the hippocampus and cerebral cortex. Ultrastructurally, some neurons demonstrated morphologic features more typical of necrosis. These studies provide evidence that apoptosis plays an important role in the pathogenesis of rabies virus infection.

Apoptosis-like cell death in experimentally-induced cryptorchidism in adult mice.

In order to elucidate the mechanism of germ cell degeneration in experimental cryptorchidism, we examined the testes of adult mice from a morphological standpoint. Adult ICR mice were made cryptorchid either unilaterally or bilaterally. In some mice, testes were surgically replaced back into the scrotum at 2 months after induction of cryptorchidism to observe the regenerative process. Morphological changes of cryptorchid and replaced testes have been studied by light and electron microscopy. Testes were also examined by the TUNEL (TdT-mediated dUTP-biotin nick end labelling) method to evaluate whether the degenerative cells, spermatocytes and spermatids, were dying by apoptosis or by any other process(es). At 8 weeks after the induction of cryptorchidism, the seminiferous epithelium consisted only of Sertoli cells, spermatogonia, and some spermatocytes of early meiotic stages. Soon after the replacement of testes to the scrotum, most of the seminiferous tubules resumed spermatogenic processes. Many degenerating cells, especially the spermatocytes, showed condensation of the nucleus and cytoplasm in cryptorchid testes. Although the cytoplasm was markedly eosinophilic under a light microscope to imply condensation of the cytoplasm, the extent of the condensation was not as pronounced under an electron microscope as reported in previous publications. The cytoplasm showed no expansion. By the TUNEL method, many of the degenerating cells, mainly the spermatocytes, have been shown as undergoing apoptosis. These data provide evidence that at least some of the cells die by apoptosis, or by a process similar to apoptosis.

Immunoadjuvant effect of inactivated Cryptococcus neoformans.

Cryptococcus neoformans disseminated into the central nervous system (CNS) of intraperitoneally inoculated adult ICR mice, but did not potentiate penetration of concurrently given Bhanja virus (Bunyaviridae) into the CNS. Likewise, Bhanja virus infection did not affect significantly the course of murine cryptococcosis. However, formalin-killed C. neoformans cells non-specifically increased production of antibodies against the virus infection in the animals.

Delta 8 desaturation in vivo of deuterated eicosatrienoic acid by mouse liver.

In vitro evidence has been reported for an alternate pathway that involves delta 8 desaturation of n-6 and n-3 polyunsaturated fatty acids (PUFA). The present study was designed to allow detection of delta 8 desaturation in vivo and to provide an estimation of the relative contribution of delta 8 desaturation to the in vivo synthesis of n-3 fatty acids. Male adult ICR mice were fed a semisynthetic fat-free diet for eight days, and then the diets were supplemented for three days with deuterated 11,14,17-eicosatrienoic acid (20:3-d8) labeled at the 3,3,4,4,8,8,9,9 carbon positions. Analysis of liver total lipid by gas chromatography/mass spectroscopy indicates that the total deuterated fatty acids contained 22.3% 20:3n-3-d8 and 28.9% of metabolites formed by elongation and delta 5 desaturation of 20:3n-3-d8. Deuterated metabolites resulting from retroconversion to 18:3-d6 and subsequent metabolism by classical pathways represented 35.3% of the total deuterated fatty acids. The retroconversion product (18:4n-3-d6) of 20:4n-3-d6 and/or -d8 was 9.0% of the total. A minor percentage (4.4%) of the products identified (20:4n-3-d6, 20:5n-3-d6, 22:5n-3-d6, 22:6n-3-d5 and 24:6n-3-d5) were formed by delta 8 desaturation. This study provides the first clear evidence of delta 8 desaturation in vivo in the mouse liver. Whether delta 8 desaturation would have a greater importance in vivo when the delta 6 desaturase pathway is disrupted remains to be determined.

Characterization of the Mode of Hantaan Virus Infection in Adult Mice Using a Nested Reverse Transcriptase Polymerase Chain Reaction: Transient Virus Replication in Adult Mice

A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse-transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)-PCR followed by nested PCR. In 5-week-old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76-118 (1.3 x 10(5) FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post-inoculation (p.i.) by nested PCR and virus-isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods. The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1:203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.

Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice.

The neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.

Systemic injections of ciliary neurotrophic factor induce sprouting by adult motor neurons.

The ability of ciliary neurotrophic factor (CNTF) to induce sprouting by undamaged adult motor neurons was studied in gluteal muscles of adult ICR mice. Low doses of CNTF (0.02 mg kg-1 day-1) only induced sprouting in gluteus muscles that were beneath the site of injection, whereas high doses of CNTF (0.4-1.2 mg kg-1 day-1) acted systemically to induce motor neuron sprouting. We found little difference between the type or quality of sprouting induced by CNTF compared with muscle paralysis. Both stimuli induced sprouts of the same length, although muscle paralysis tended to induce more sprouts per end-plate. Paralysis also induced more nodal sprouting than did CNTF, but both were weaker stimuli for nodal sprouting than was partial denervation. In addition to its effects on motor neuron sprouting, high doses of CNTF induced loss of up to 36% of the body weight of treated mice. The substantial wasting caused by CNTF indicates that the factor has potent cachectic activity.

Rapid freezing replica studies of membrane specializations in the proximal tubules of mouse kidney.

The membrane specializations of the fresh unfixed kidney cortex of adult and neonatal ICR mice were examined by using rapid freezing replica methods. In proximal tubular cells, numerous apical intracellular tubules exhibited helical patterns on the E face with a pitch of about 12 nm. This regular pattern was often continuous with similar striped indentations on the edge of the vacuoles connecting with the tubules. On the luminal surface (ES) of these vacuoles, membrane surface particles were arranged regularly in striped patterns with a center-to-center spacing of about 12 nm. We could not identify differentiations on the PF or PS of the same membrane systems. Another membrane specialization was a plaque or patch of clear pits in tilted lattice alignments on the P face of the large vacuoles with a center-to-center spacing of about 20 nm. This type of specialization was often observed in the neonatal mice proximal tubular cells. These membrane specializations may indicate the active membrane functions in the proximal tubules and suggest the functional continuity and structural relationship of these apical endocytic membrane systems.

The pathogenicity of ribonucleotide reductase-null mutants of herpes simplex virus type 1 in mice.

The pathogenicity of ribonucleotide reductase (RR)-null mutants (hrR3 and ICP6 delta) of herpes simplex virus (HSV) type 1 was studied after intracerebral and corneal inoculation in newborn and adult ICR mice. ICP6 delta failed to replicate in brains of mice greater than or equal to 8 days old but exhibited significant virulence for newborn mice as a result of viral replication in the brains. The RR- and a thymidine kinase (TK)-deficient mutant of HSV-1 strain KOS could grow in eye tissues of adult ICR mice. Viral DNA of hrR3 was detected in brain tissues of intracerebrally infected mice or in the trigeminal ganglia of corneally infected mice greater than or equal to 50 days after infection, and infectious hrR3 could be recovered from these tissues by superinfection of the mice with wild-type HSV-2. These observations indicate that pathogenicity of RR- mutants in mice is highly dependent on the physiologic state of tissues infected and that RR- mutants have the ability to establish latency in nervous system tissues of mice by either the peripheral or intracerebral route. It was also demonstrated that the inability of the RR- mutants to invade the central nervous system was efficiently complemented by simultaneous infection with another defective virus, the TK- mutant of KOS.

Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.