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PubMed2005

Improvement on genetic transformation in the nematode-trapping fungus Arthrobotrys oligospora and its quantification on dung samples.

An improved DNA-mediated transformation system for nematode-trapping fungus Arthrobotrys oligospora based on hygromycin B resistance was developed. The transformation frequency varied between 34 and 175 transformants per microg linearized DNA and 93% of the transformants were stable for drug resistance when tested 100 randomly selected transformants. More than 2000 transformants were obtained by transformation of the fungus with pBChygro in the presence of HindIII and among them, one, YMF1.00110, which lost its ability of forming predacious structure, was isolated. Southern analysis showed that the plasmid DNA had integrated into the genome of all tested transformants (including YMF 1.00110) except one. The transformant tagged with hph gene could be re-isolated and quantified from dung samples based on the resistance of hygromycin B. All the results suggested that the method of restriction enzyme mediated integration (REMI) should facilitate not only the insertional mutagenesis for tagging and analysis genes of interest but also the ecological investigation of tagged fungi in a given environment.

Animals↗
PubMed1989

Genetic transformation of mouse cultured cells with the help of high-velocity mechanical DNA injection.

NIH 3T3 mouse cells were transfected by the plasmid pSV3neo (G418-resistant) with the help of high-velocity mechanical DNA injection based on the principle of bombarding cells with tungsten particles covered with the DNA. Stable transformants were obtained. Dot-hybridization and Southern analysis revealed the integration into the genome of 5-20 copies per cell of original plasmid DNA. The plasmid DNA was shown to have tandem organization.

Animals↗
PubMed1991

Genetic transformation of Aureobasidium pullulans.

Aureobasidium pullulans strain Y117 was transformed to hygromycin resistance using plasmid pDH33, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Aspergillus niger glucoamylase gene (glaA). Southern hybridizations of transformants revealed multiple, integrated copies of the vector. The glaA promoter was not induced by starch in A. pullulans as it is in A. niger; however, the transcriptional start points were the same in both species.

Aspergillus niger↗
PubMed1996

Genetic transformation of Vitreoscilla sp.

Of all the methods customarily used to transform E. coli we found only electroporation to be effective for transformation of the Gram-negative bacterium Vitreoscilla, yielding 5.10(5) transformants/microgram of plasmid DNA. The conditions used were close to those described for E. coli E. coli plasmids are stably maintained in Vitreoscilla. This is the first report of exogenous DNA transfer in Vitreoscilla which opens the way for the application of recombinant-DNA techniques to study this unique group of organisms.

Electroporation↗
PubMed2004

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana.

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.

Agrobacterium tumefaciens↗
PubMed1985

Genetic transformation of the phytopathogenic bacteria, Erwinia chrysanthemi.

Erwinia chrysanthemi is an enterobacterium whose phytopathogenicity is due to its pectinolytic and cellulolytic activities. The CaCl2 mediated transformation procedure was successfully applied to two E. chrysanthemi wild type strains. The highest efficiency of transformation of E. chrysanthemi with pBR322 was found using 0.1 M CaCl2, 0.1 M MgCl2 treated cells and a heat pulse at 30 degrees C for 6 min. This yielded about 600 transformants per microgram of pBR322 DNA and 2.3 X 10(-6) per viable cell. Plasmid stability after twenty generations was lower than in E. coli: only 40-60% of the cells retained the plasmid in the absence of selective pressure. Based on this result, cloning in E. chrysanthemi with pBR322 vectors should therefore be possible, making it a potential host for cloning any gene for biomedical or industrial purposes.

Calcium Chloride↗