High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.

The use of transfection in the study of the biology of malaria parasites has been limited due to poor transfection efficiencies (frequency of 10(-6) to 10(-9)) and a paucity of selection markers. Here, a new method of transfection, using non-viral Nucleofector technology, is described for the rodent parasite Plasmodium berghei. The transfection efficiency obtained (episomal and targeted integration into the genome) is in the range of 10(-2) to 10(-3). Such high transfection efficiency strongly reduces the time, number of laboratory animals and amount of materials required to generate transfected parasites. Moreover, it allows different experimental strategies for reverse genetics to be developed and we demonstrate direct selection of stably and non-reversibly transformed, fluorescent protein (FP)-expressing parasites using FACS. Since there is no need to use a drug-selectable marker, this method increases the (low) number of selectable markers available for transformation of P. berghei and can in principle be extended to utilise additional FP. Furthermore the FACS-selected, FP-expressing parasites may serve as easily visualized reference lines that may still be genetically manipulated with the existing drug-selectable markers. The combination of enhanced transfection efficiency and a versatile rodent model provides a basis for the further development of novel tools for high throughput genome manipulation.

Efficient transfection of primary cells relevant for cardiovascular research by nucleofection.

Cell types that are important for cardiovascular research, e.g., cardiomyocytes, endothelial cells, or adult stem cells, are often hard to isolate, culture, and transfect. Low-transfection efficiencies are a major limitation because, in many cases, results achieved with surrogate model cell lines, if any at all are available for the primary cell type of interest, do not reflect the situation in the primary cell. We have demonstrated that unprecedented transfection results are achieved with primary cells when novel electroporation conditions are combined with a treatment of the cells in specific solutions that help stabilize the cells in the electrical field. This led to the development of the new proprietary transfection technology nucleofection. Nucleofection has proved to be successfully applicable to a variety of primary cells and other hard-to-transfect cell lines, and, thus, opens unique perspectives for novel experimental setups as therapeutic strategies. Herein we present protocols for the efficient nucleofection of human umbilical vein endothelial cells, human coronary artery endothelial cells, smooth muscle cells (e.g., pig vascular smooth muscle cells), neonatal rat cardiomyocytes, and human mesenchymal stem cells and depict some results obtained with such transfected cells.

Rapid and highly efficient gene transfer into natural killer cells by nucleofection.

Natural killer (NK) cells are important mediators of virus- and tumor-specific immune responses. The transfection of genes into NK cells has been proven difficult and so far requires infection with virus-based vectors. Here, the application of a novel nonviral, electroporation-based gene transfer method is described for the rapid and highly efficient transient transfection of NK cell lines as well as freshly isolated NK cells. In contrast to conventional methods, this technique, termed nucleofection, leads to direct transfer of DNA into the nucleus. Using reporter proteins H-2K(k), luciferase+, and enhanced yellow green fluorescent protein (EYFP) as independent read-out systems, transfection efficiencies of well over 50% were achieved in transient transfection assays. The highest luciferase activity could be measured only 4 h after transfection, whereas EYFP, when analyzed by flow cytometry, showed expression peaks after 28 h. Interestingly, best transfection efficiencies were achieved with non-dividing NK cells. The novel nuclear gene transfer method presented here is highly useful for the analysis of NK cell-specific gene regulation and should facilitate the development of NK cell-based gene therapy approaches.

Rapid and efficient electroporation-based gene transfer into primary dissociated neurons.

Non-viral gene transfer into neurons has proved to be a formidable task. Here, we describe an electroporation-based method that allows efficient and reliable DNA transfer into dissociated neural cells before they are plated and cultured. In hippocampal neural cells derived from either neonatal mouse or embryonic chicken brains, a high transfection rate was already observed 5 h after transfection, and reached 40-80% in 24 h, as monitored by expression of enhanced green fluorescent protein (eGFP). The level of eGFP expression per cell depended on the amount of DNA used in a gene transfer experiment. The survival and neuritic length of transfected cells resembled that of non-electroporated cells. The transfected neurons showed normal immunostaining for endogenous synaptic protein synaptophysin and the neural cell adhesion molecule (NCAM). Furthermore, efficient gene transfer of the NCAM isoform NCAM140 and eGFP-tagged NCAM140 could be achieved, allowing visualization of NCAM140 expression. Also, a glycosylphosphatidylinositol-anchored eGFP could be efficiently expressed, highlighting lipid rafts without altering electrophysiological properties of transfected neurons. When neurons transfected with green and red fluorescent proteins were cocultured, fine details of their interactions could be revealed in time-lapse experiments. Thus, the method provides a useful tool for elucidation of genes involved in different neuronal functions, including neurite outgrowth, synaptogenesis and synaptic transmission.

alpha-Tropomyosin mutations Asp(175)Asn and Glu(180)Gly affect cardiac function in transgenic rats in different ways.

To study the mechanisms by which missense mutations in alpha-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing alpha-tropomyosin with one of two disease-causing mutations, Asp(175)Asn or Glu(180)Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-alpha-tropomyosin antibody, ARG1. In transgenic rats with either alpha-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca(2+) sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp(175)Asn, but not Glu(180)Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca(2+) waves were detected only in cardiomyocytes from animals with mutation Asp(175)Asn, suggesting an increase in intracellular Ca(2+) concentration compensating for the reduced Ca(2+) sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp(175)Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by alpha-tropomyosin mutation Asp(175)Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca(2+) handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.