Methods in molecular biology
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As the field of proteomics shifts from qualitative identification of protein "subfractions" to quantitative comparison of proteins from complex biological samples, it is apparent that the number of approaches for quantitation can be daunting for the result-oriented biologist. There have been many recent reviews on quantitative proteomic approaches, discussing the strengths and limitations of each. ⋯ Here we present a detailed bioinformatics workflow for one of the simplest, most pervasive quantitative approach-spectral counting. The informatics and statistical workflow detailed here includes newly available freeware, such as SePro and PatternLab which post-process data according to false discovery rate parameters, and statistically model the data to detect differences and trends.
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Experimental spinal cord injury (SCI) can maintain the continuity of the spinal cord, as in the contusion (e.g., weight-fall) or compression models, or not, when there is a partial or a complete transection. The majority of acute human SCI is not followed by complete transection, but there is a combination of contusion, compression, and possibly partial transection. ⋯ This lesion was established by our group and represents a simple, reliable, and inexpensive clip compression model with functional and morphological reproducibility. In this chapter we describe, step by step, the protocol of this experimental SCI.
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In animals, environmental exposure such as toxic chemicals and microorganisms or pathophysiological conditions in respiratory system could result in inflammatory response in their lungs. Bronchoalveolar lavage (BAL) is a procedure that can be used to collect samples from animal lungs to efficiently evaluate the immune response by examining both the compositions of cells and fluid from lavage. The profile of inflammatory cells in BAL provides a qualitative description of inflammatory response and the secretion in BAL fluid contains proteins of inflammatory mediators and albumin as a quantitative measurement of inflammation and tissue injury in the lungs. A consistent experimental approach on how to lavage mouse lungs and collect samples is important for a reproducible evaluation of pathological and physiological changes in mouse lung especially for the analysis of inflammation.
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The liver is a very complex organ with a large variety of functions, making it an attractive organ for gene replacement therapy. Many genetic disorders can be corrected by delivering gene products directly into the liver using viral vectors. In this chapter, we will describe gene delivery via portal vein administration in mice and dogs to correct the blood coagulation disorder hemophilia B. ⋯ Complete correction of murine hemophilia B and multi-year near-correction of canine hemophilia B have been achieved following portal vein delivery of adeno-associated viral (AAV) vectors expressing factor IX from hepatocyte-specific promoters. Peripheral vein injection can lead to increased vector dissemination to off-target organ such as the lung and spleen. Below, we will describe portal vein injection delivery route via laparotomy.
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Irreversible electroporation has recently been applied to tissue and tumor ablation. This animal model was developed to evaluate optimal parameters for subcutaneous tumor ablation. ⋯ Under general anesthesia with complete muscle relaxation, electroporation was performed with the NanoKnife device. Post-procedure tumors were recovered and studied at specified time intervals to assess the efficacy.