Mouse Tail Tissue Research Articles

Anti-tumour effect of neo-antigen-reactive T cells induced by RNA mutanome vaccine in mouse lung cancer

PurposeMutation-specific T-cell response to epithelial cancers and T-cell-based immunotherapy has been successfully used to treat several human solid cancers. We aimed to investigate the anti-tumour effect of neo-antigen-reactive T(NRT) cells induced by RNA mutanome vaccine, which may serve as a feasible and effective therapeutic approach for lung cancer.MethodsWe predicted candidate neo-antigens according to the mutant gene analysis by sequencing the mouse Lewis cells and C57BL/6 mouse tail tissue. RNA vaccine was prepared with the neo-antigens as the template. We assessed antitumor efficacy, cytokine secretion and pathological changes after adoptive transfer of NRT cells in vitro and vivo experiments.ResultsWe identified 10 non-synonymous somatic mutations and successfully generated NRT cells. The percentage of T-cell activation proportion was increased from 0.072% in conventional T cells to 9.96% in NRT cells. Interferon-γ secretion augmented from 17.8 to 24.2% as well. As an in vivo model, adoptive NRT cell infusion could promote active T-cell infiltration into the tumour tissue and could delay tumour progression.ConclusionNRT cells induced by RNA mutanome vaccine exert a significant anti-tumour effect in mouse lung cancer, and adoptive NRT cell therapy might be considered a feasible, effective therapeutic approach for lung cancer.

Preparation of Polymerase Chain Reaction Template DNA from Mouse Tail Tissue.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). In this protocol, a small piece from the tip of the tail is removed and processed using hot sodium hydroxide and Tris (HotSHOT).

Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T.tricuspidata. Further, purified T.tricuspidata DNase showed a single sharp peak in reversed-phase high-performanceliquid chromatography(RP-HPLC) with an apparent molecular mass of 17 kDa. T.tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T.tricuspidata was able to neutralize E.carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T.tricuspidata DNase was also able to reverse E.carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T.tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T.tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.

Longitudinal effects of developmental bisphenol A and variable diet exposures on epigenetic drift in mice

Environmental factors, including exogenous exposures and nutritional status, can affect DNA methylation across the epigenome, but effects of exposures on age-dependent epigenetic drift remain unclear. Here, we tested the hypothesis that early-life exposure to bisphenol A (BPA) and/or variable diet results in altered epigenetic drift, as measured longitudinally via target loci methylation in paired mouse tail tissue (3 wks/10 mos old). Methylation was quantified at two repetitive elements (LINE-1, IAP), two imprinted genes (Igf2, H19), and one non-imprinted gene (Esr1) in isogenic mice developmentally exposed to Control, Control+BPA (50μg/kg diet), Mediterranean, Western, Mediterranean+BPA, or Western+BPA diets. Across age, methylation levels significantly (p<0.050) decreased at LINE-1, IAP, and H19, and increased at Esr1. Igf2 demonstrated Western-specific changes in early-life methylation (p=0.027), and IAP showed marginal negative modification of drift in Western (p=0.058) and Western+BPA (p=0.051). Thus, DNA methylation drifts across age, and developmental nutritional exposures can alter age-related methylation patterns.

Development of a cellular biosensor system for genotoxicity detection based on Trp53 promoter.

To develop a mouse cell biosensor system for the high-throughput genotoxicity detection of chemicals, such as environmental pollutants. We developed a novel reporter vector pGL4-GFP, wherein the firefly luciferase reporter gene in the pGL4.82 vector was replaced by the green fluorescent protein (GFP) gene from the pAcGFP1-N1 vector. To construct the reporter pGL4-p53-GFP (p53 promoter linked to GFP), a fragment containing the p53 gene promoter was generated by amplifying a region from -481 to +180 of mouse genomic DNA isolated from mouse tail tissue. We developed a mouse cell biosensor system for the high-throughput genotoxicity detection of new drugs by stably integrating the reporter plasmid of pGL4-p53-GFP into the mouse embryonic fibroblast cells. Various genotoxic agents were used to treat this biosensor system. The resulting fluorescence was directly observed under a fluorescence microscope, and the GFP protein level was measured through Western blot analysis. The biosensor system was treated with genotoxic agents, such as doxorubicin, cyclophosphamide, and benzo(a)pyrene. The GFP protein expression was significantly increased in cells exposed to genotoxic agents but negatively responded to the non-genotoxic agent dimethyl sulfoxide, thereby proving the specificity and sensitivity of the biosensor system. This novel in vitro biosensor system can be especially useful in genotoxicity detection.

Evaluation of a reliable and cost-effective method of DNA isolation for mouse genotyping

Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, "crude" DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.

Imaging with second-harmonic radiation probes in living tissue

We demonstrate that second-harmonic radiation imaging probes are efficient biomarkers for imaging in living tissue. We show that 100 nm and 300 nm BaTiO3 nanoparticles used as contrast markers could be detected through 50 μm and 120 μm of mouse tail tissue in vitro or in vivo. Experimental results and Monte-Carlo simulations are in good agreement.

Generation of Transgenic Mice Overexpressing a Hybrid Enzyme Up‐regulating Biosynthesis of Vascular Protector Prostacyclin

Prostacyclin (PGI2) is a potent vascular protector through its anti‐platelet aggregation and vasodilation properties. Non‐steroid anti‐inflammatory drugs (NSAIDs) by inhibiting PGI2 production promote heart diseases. Recently, we have successfully engineered a hybrid enzyme (COX‐1‐10aa‐PGIS, by linking cyclooxygenase isoform 1(COX‐1) with PGI2 synthase (PGIS) through a 10 amino acids (10aa) linker) with triple catalytic functions directly converting arachidonic acid into PGI2. PGI2 biosynthesis was upregulated in the mammalian cells, transfected with the hybrid enzyme cDNA. Thus, with the objective of translating these in vitro results into in vivo, we generated transgenic mice overexpressing COX‐1‐10aa‐PGIS. PCR using mouse tail tissue confirmed the incorporation of COX‐1‐10aa‐PGIS gene in the transgenic mouse chromosome. Permanent expression of the hybrid enzyme was identified by Western blot. The catalytic activity of the hybrid enzyme to form PGI2 was determined by enzyme activity assay and LC/MS analysis of transgenic mouse tail and urine, respectively. In conclusion, a transgenic mouse overexpressing the hybrid enzyme with upregulated PGI2 biosynthesis was successfully obtained. The mice can be used to further uncover the mechanisms of the vascular protection of PGI2 and test the side effects of NSAIDs.Funding: R01 HL56712 (for KHR); RO1HL079389 (for KHR) and RC1 HL100807 (for RD and KHR)

IDQC MRI weighted by longitudinal relaxation in the rotating frame

A long-duration, low-power, off-resonance spin-locking pulse was incorporated into the COSY revamped by asymmetric z gradient-echo detection (CRAZED) pulse sequence in order to evaluate the effects of intermolecular double-quantum longitudinal relaxation in the tilted rotating frame (T1rho,DQ(eff)). This modified CRAZED sequence was followed by a standard fast spin-echo imaging sequence to form images with T1rho,DQ(eff)-weighted contrast. Imaging experiments were performed on an agarose-gel phantom and mouse-tail tissue at 600 MHz. Experimental results demonstrated the feasibility of imaging applications based on T1rho,DQ(eff) as a novel contrast mechanism, and showed that iDQC off-resonance longitudinal relaxation in the rotating frame T1rho,DQ(eff) is sensitive to the tilt angle theta and the effective spin-locking field omegae. Imaging based on T1rho,DQ(eff)) has reduced RF power deposition compared to on-resonance spin-locking, which is advantageous for human applications.

A Procedure for Culture of Cells From Mouse Tail Biopsies: &lt;italic&gt;Brief Communication&lt;/italic&gt;&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

A simple technique was developed for production of cell cultures from mouse tails. The use of mouse tail tissue for biopsies made repeated sampling of animals possible without their health or breeding performance being jeopardized. The procedure proved to be particularly useful in studies of murine leukemia viruses, because it allowed virus induction and Fv-1 gene-typing tests to the done on biopsy tissue from adult animals.

In vivo NMR relaxation studies of tumors

Pulsed nuclear magnetic resonance has been used to differentiate between normal mouse tail tissue and a malignant transplanted melanoma S91 located on the tail of a live mouse. Measurements of proton spin-lattice and spin-spin relaxation serve to detect and monitor the tumor growth. The mature tumor exhibits a spin-lattice relaxation time T <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</inf> of <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">\sim0.7- 0.8</tex> s contrasting with the corresponding normal tail tissue T <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</inf> of half this value. On the other hand spin-spin relaxation in normal tissue cannot be characterized by a single relaxation time T <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</inf> . The corresponding relaxation in a mature tumor is found to be closer to a single exponential but still requires at least two superimposed exponential decays.

Recognition of cancer in vivo by nuclear magnetic resonance.

Pulsed nuclear magnetic resonance has been used to differentiate in vivo between normal mouse tail tissue and a malignant transplanted melanoma, S91, located on the tail. The tumor displayed a nuclear (proton) spin-lattice relaxation time of approximately 0.7 second contrasted with the simultaneously measured normal tail tissue relaxation time of approximately 0.3 second.