We developed a simple microtechnique to measure lipids in milk by UV spectrophotometry. This technique is based upon the property of fatty acids to absorb UV light proportional to their concentration. Samples of powdered or fluid milk (30 or 60 muL) were added to 3 mL of analytic grade ethanol and stored at -20 degrees C for at least 1 h. This procedure precipitates proteins and hydrophobic peptides that interfere with UV measurement. Sample absorbances are then measured at 208 nm in an UV-Vis spectrophotometer. This technique correlated very well against Milko-Scan, a device that measures milk fat by IR spectroscopy, with an r(2) >0.982. Accuracy and precision, evaluated by recovery and replicate assays, are also very acceptable. This method is suitable as a fast, cost-effective alternative screening method to estimate milk fat content in small samples without prior lipid extraction.
Transgenic farm animals are attractive alternative mammalian models to rodents for the study of developmental, genetic, reproductive and disease-related biological questions, as well for the production of recombinant proteins, or the assessment of xenotransplants for human patients. Until recently, the ability to generate transgenic farm animals relied on methods of passive transgenesis. In recent years, significant improvements have been made to introduce and apply active techniques of transgenesis and genetic engineering in these species. These new approaches dramatically enhance the ease and speed with which livestock species can be genetically modified, and allow to performing precise genetic modifications. This paper provides a synopsis of enzyme-mediated genetic engineering in livestock species covering the early attempts employing naturally occurring DNA-modifying proteins to recent approaches working with tailored enzymatic systems.
Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co-injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase-catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid-based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi-transposon transgenic mice in a single step.
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