Baker´s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

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Candidate DNA vaccines for hemorrhagic fever with renal syndrome expressing the envelope glycoprotein genes of Hantaan (HTNV) or Puumala (PUUV) viruses were evaluated in an open-label, single-center Phase 1 study consisting of three vaccination groups of nine volunteers. The volunteers were vaccinated by particle-mediated epidermal delivery (PMED) three times at four-week intervals with the HTNV DNA vaccine, the PUUV DNA vaccine or both vaccines. At each dosing, the volunteers received 8 μg DNA/4 mg gold. There were no study-related serious adverse events, and all injection site pain was graded as mild. The most commonly reported systemic adverse events were fatigue, headache, malaise, myalgia, and lymphadenopathy. Blood samples were collected on days 0, 28, 56, 84, 140, and 180, and assayed for the presence of neutralizing antibodies. In the single vaccine groups, neutralizing antibodies to HTNV or PUUV were detected in 30% or 44% of individuals, respectively. In the combined vaccine group, 56% of the volunteers developed neutralizing antibodies to one or both viruses. These results demonstrate that the HTNV and PUUV DNA vaccines are safe and can be immunogenic in humans when delivered by PMED.

OBJECTIVE: To establish a preparation method of cartridge for gene gun and different gene transfection to MVF-7 cell lines in vitro.
METHODS: The cartridge was prepared by precipitation method of spermidine and calcium chloride.using gene gun method, respectively, the eukaryotic expression plasmid pEGFP, Pmcherry transfected with the control group and experimental group MCF-7 cells 24 h after transfection.
RESULTS: The preparation of the gene-gun bullets, gene gun-mediated pEGFP, Pmcherry be able to separate and co-transfected into cultured MCF-7 cells, 24 h after transfection could be detected in red, green fluorescence, while the control group there was no fluorescent protein expression.
CONCLUSIONS: Gene gun can be effectively mediated gene transfer and be able to achieve a common transfer of two genes. The report gene can be expressed through gene gun-transfected MCF-7 cells.

Base substitutions equivalent to those causing human pathologies have been introduced in yeast mitochondrial tRNA genes. These mutants can be utilized as flexible tools to investigate the molecular aspects of mitochondrial diseases and identify correcting genes. We show that for all studied tRNA mutations (including an homoplasmic one in tRNA(Val)) the severity of phenotypes follows the same trend in four different nuclear backgrounds. Correcting genes include TUF1 and genes encoding aminoacyl-tRNA synthetase. The effect of suppressors was analyzed by Northern blot. Mutated leucyl-tRNA synthetase with highly reduced catalytic activity maintains full suppressing effect, thus suggesting a chaperone-like and/or stabilizing function.

One of the key elements concerning our understanding of the organization of the mouse retina is the complete classification of the various types of bipolar cells. With the present study, we tried to contribute to this important issue. Unfortunately, most of the antibodies that stain specifically bipolar cells in the retina of other mammals hardly work for the retina of the mouse. We succeeded in overcoming this limitation by using a relatively novel technique based on the gene gun transfer of fluorescent dyes to cells. Hence, we were able to stain a considerable number of bipolar cells that could be characterized according to morphological and comparative criteria. We also performed a complete morphometric analysis of a subset of bipolar cells stained by anti-neurokinin-3 receptor antibodies. We found nine types of cone bipolar cells and one type of rod bipolar cell; these data are consistent with the findings of previous studies on the retinas of other mammals, such as rabbits, rats, and monkeys and with a recent study based on the mouse retina (Ghosh et al. [2004] J Comp Neurol 469:70-82). Our results also confirm the existence of a common structural similarity among mammalian retinas. It remains to be elucidated what is exactly the functional role of the various types of cone bipolar cells and what is the specific contribution they provide to the perception of a given visual stimulus. Most probably, each bipolar cell type constitutes a specialized channel for the computation of a selected component of the visual stimulus. More complex signal coding, involving the coordinated activity of various types of bipolar cells, could also be postulated, as it has been shown for ganglion cells (Meister [1996] Proc Natl Acad Sci U S A 93:609-614).

It was reported in this article that the transgenic elite indica container line D297B containing snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene gna was obtained by biolisties. PCR, Southern blotting and Western blotting indicated that the transgenes were integrated into the genome of D297B and expressed in transgenic plants. Analysis of protein activity showed that product of transgene had activity of agglutinin. Resistance of transgenic seeds to Hygromycin B suggested that the transgenes were integrated in a single locus and inherited according to the pattern of 3:1 in most of transgenic plants. In addition, the PCR analysis revealed that the insect-resistant gene gna and selective marker gene hpt were co-integrated and co-inherited.

The primary objective of this phase I study was to determine the safety of an autologous tumor vaccine given by intradermal injection of lethally irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transfected autologous melanoma and sarcoma cells. Secondary objectives included validation of the gene delivery technology (particle-mediated gene transfer), determining the host immune response to the tumor after vaccination, and monitoring patients for evidence of antitumor response. Sixteen patients were treated with either of two different doses of GM-CSF-treated tumor cells. One patient received treatment with both doses of tumor cells. No treatment-related local or systemic toxicity was noted in any patient. Patients administered 100% treated cells (i.e., with a preparation of tumor cells that had all been exposed to GM-CSF DNA transfection) had a more extensive lymphocytic infiltrate at the vaccine site than did patients given 10% treated cells (a preparation of tumor cells in which 10% had been exposed to GM-CSF transfection) or nontreated tumor. The generation of a systemic immune response to autologous tumor by a delayed-type hypersensitivity response to the intradermal placement of nontransfected tumor cells was noted in one patient. One patient had a transient partial response of metastatic tumor sites. The entire procedure, from tumor removal to vaccine placement, was accomplished in less than 6 hr in all patients. Four of 17 patient tumor preparations produced greater than 3.0 ng of GM-CSF per 10(6) cells per 24 hr in vitro. The one patient with greater than 30 ng of GM-CSF per 10(6) cells per 24 hr in vitro had positive DTH, a significant histologic inflammatory response, and clinically stable disease. This technique of gene transfer was safe and feasible, but resulted in clinically relevant levels of gene expression in only a minority of patients.