Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transformation efficiency”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed2006

Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens.

AIMS: To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies. METHODS AND RESULTS: Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58.3 +/- 18.5, 98.3 +/- 24.8 and 169.7 +/- 35.5 (+/-SEM) transformants per 10(5), 10(6) and 10(7) target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA. CONCLUSIONS: In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.

Agrobacterium tumefaciens↗
PubMed1989

Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation.

We have undertaken a systematic study to test the transformation of various species of gram-negative bacteria using the electroporation method. The data obtained show very clearly that a great variety of gram-negative bacteria--15 different species belonging to 11 different genera--including freshly isolated wild-type strains can be transformed efficiently by use of the electric-field mediated transformation technique. These include species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, photosynthetic bacteria and strains for which transformation could not be achieved, up to now, by other methods.

Electricity↗
PubMed1985

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast.

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.

Cloning, Molecular↗
PubMed2003

Comparison of different transformation methods for Aspergillus giganteus.

Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus, the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens-mediated transformation. Electroporation and biolistic transformation were found to be inappropriate for transforming A. giganteus, due to a low transformation yield. The conventional transformation technique based on protoplasts yielded up to 55 transformants in 10(8) protoplasts/microg DNA and was enhanced to 140-fold by A. tumefaciens-mediated transfer of its T-DNA. Here, the germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency. Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.

Aspergillus↗
PubMed2005

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation.

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.

Aminobutyrates↗
PubMed1990

Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance.

To provide a dominant selectable marker for transformation of Neurospora crassa strains lacking specific auxotrophic mutations, we have engineered the bleomycin (Bm) resistance-encoding gene (ble) from the bacterial transposon Tn5 for expression in N. crassa. The coding region of the ble gene was fused to the promoter and terminator regions of the N. crassa am gene. In some vectors, multiple cloning sites were placed flanking the ble gene to provide a versatile ble cassette. When introduced into N. crassa, the hybrid ble gene conferred resistance to greater than 15 micrograms Bm/ml. Under optimal conditions, the levels of Bm required (2.5 micrograms/ml) make even large-scale transformation experiments very economical. Aspergillus nidulans could also be efficiently transformed to Bm resistance using the N. crassa ble gene fusion. Since the ble gene functions in both N. crassa and A. nidulans, the gene should be useful as a transformation marker for the many other filamentous fungi which are sensitive to Bm.

Aspergillus nidulans↗
PubMed2000

Agrobacterium-mediated sorghum transformation.

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a 'Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

Agrobacterium tumefaciens↗
PubMed1997

Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.

Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.

Animals↗
PubMed2000

[Screening of acrystalliferous mutants from Bacillus thuringiensis and their transformation properties].

After elevating growth temperature of YBT-1463 and other 8 Bacillus thuringiensis parental strains to 42 degrees C, 9 acrystalliferous(Cry-) mutants were obtained, and then a series of partial plasmid-cured mutants were further obtained from 3 Cry- mutants BMB151, BMB154 and BMB160 by elevating temperature to 44 degrees C. One of which, BMB170, was further treated by elevating temperature to 46 degrees C and adding 0.05% SDS as the plasmid-curing agent. The resulting mutant, which was designated BMB171 and had no resident plasmid, was finally obtained. The studies on the transformation efficiency and stability of Cry- mutants showed that the transformation frequencies, as well as stability of several exogenous plasmids(pHT3101, pBMB1736, etc.) introduced into Cry- mutants by electroporation, were correlated with the numbers of resident plasmids bearing by the relevant recipients, the patterns of plasmid replicons and the sizes of those exogenous plasmids. Meanwhile, a much higher electro-transformation frequencies of Cry- mutants than the parental strain YBT-1463 was found, and the highest of the transformation frequency, in BMB 171, could reach up to 10(7) transformants/microgram DNA, and the stability of those exogenous plasmids in BMB171 were also higher than in other Cry- mutants and YBT-1463 strain.

Bacillus thuringiensis↗
PubMed1991

Highly transformable mutants of Streptomyces aureofaciens containing restriction-modification systems.

Streptomyces aureofaciens 13 is a mutant defective in chlortetracycline production. It was chosen as a potentially useful host for gene cloning in investigations of the organization of the biosynthetic genes for the tetracycline antibiotic pathway. From the Streptomyces aureofaciens 13 strain, three suitable clones were used for our work. The conditions for optimal formation and efficient transformation of protoplasts with plasmid DNAs have been determined. Transformation frequencies of about 10(4) to 10(5) per microgram of plasmid DNA were obtained when plasmids were isolated from Streptomyces strains. From the patterns of restriction enzyme digestion of plasmid DNA isolated from Streptomyces aureofaciens transformants, it was observed that the clones express modification systems which render plasmid DNAs resistant to cleavage by HindIII and EcoRI. Additionally, one of the clones produces the restriction endonuclease Sau13I (isoschizomer of SauI). The presence of the restriction-modification system of Sau13I does not reduce the efficiency of plasmid transformation.

DNA Restriction Enzymes↗
PubMed2004

High-frequency transformation of Lobelia erinus L. by Agrobacterium-mediated gene transfer.

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a beta-glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3-4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high--45% per inoculated disc.

Agrobacterium tumefaciens↗
PubMed2003

Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker.

To generate transgenic planarians we used a set of versatile vectors for animal transgenesis based on the promiscuous transposons, mariner, Hermes and piggyBac, and a universal enhanced GFP (EGFP) marker system with three Pax6 dimeric binding sites, the 3xP3-EGFP developed by Berghammer et al. [Berghammer, A. J., Klinger, M. & Wimmer, E. A. (1999) Nature 402, 370-371]. This marker is expressed specifically in the eyes of various arthropod taxa. Upon microinjection into the parenchyma of adult planarians and subsequent electroporation, these vectors transpose efficiently into the planarian genome. One of the cell types transformed are the totipotent "neoblast" stem cells present in the adults, representing 30% of total cells. The neoblast represents a unique cell type with the capacity to proliferate and to differentiate into all somatic cell types as well as into germ cells. All three transposon vectors have high transformation efficiency, but only Hermes and piggyBac show stable integration. The mariner vector is frequently lost presumably because of the presence of active mariner-type transposons in the genome of the Girardia tigrina. Transformed animals are mosaics containing both transformed and untransformed neoblasts. These differentiate to form EGFP-positive and -negative photoreceptor cells. Such mosaicism is maintained through several cycles of regeneration induced by decapitation or asexual reproduction. Transformed neoblasts also contribute to the germ line, and can give rise to pure transgenic planarian lines in which EGFP is expressed in all photoreceptor cells after sexual reproduction. The presence of the transgenes was confirmed by PCR, plasmid rescue assay, inverse PCR, and Southern blotting. Our results with the 3xP3-EGFP marker confirm the presence of Pax6 activity in the differentiated photoreceptor cells of planarian eyes. Transgenesis will be an important tool to dissect developmental molecular mechanisms in planarian regeneration, development and stem cell biology, and may also be an entry point to analyze the biology of parasitic Platyhelminthes.

Animals↗
PubMed2004

A rapid and highly efficient method for transformation of sugarcane callus.

Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult. Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars. One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol. An improved protocol for Agrobacterium-mediated transformation of sugarcane is described. Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene. The whole procedure permitted the production of transgenic calli in a short time (1.5 mo). The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants. This protocol may be implemented also for the generation of transgenic plants from other species.

6-Phytase↗
PubMed2003

[Protoplast isolation and transformation of Exserohilum turcicum].

Northern corn leaf blight, caused by the fungus Exserohirum turcicum Pass. (Leonard and Suggs), is one of the major diseases in most corn-growing areas of the world. Research on gene tagging of E. turcicum has been limited due to the lack of an efficient transformation system. Since E. turcicum produces and accumulates melamin in cell walls during vegetative growth, it is difficult to efficiently isolate its protoplast. To isolate the protoplast of this pathogen with a high frequency, the effects of cell wall degradation enzymes, including beta-1,3-glucanase (Fungase, Funcelase, Novozyme and Glucanex) and beta-glucuronidase (Driselase, Uskizyme and Kitalase), enzyme concentrations, combinations, strains and medium on the isolation frequency were tested. The isolation frequencies were high enough for transformation when the combinations of (Kitalase + Glucanex + Driselase), (Kitalase + Glucanex) or (Kitalase + Uskizyme) were used. Moreover, the isolation frequencies of protoplast were significantly affected by the cultural morphologies of strain and the growth stage of mycelia. Among the plasmids tested, only plasmid pAN71 is efficient for transformation of E. turcicum. This result will provide some useful information for gene tagging of E. turcicum and other species in Exserohirum.

Ascomycota↗
PubMed1986

A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state.

A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids.

Animals↗
PubMed1992

Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae.

Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast replicating plasmids, carrying the HIS3, TRP1, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces cerevisiae cells. Repair was assayed as the relative transformation efficiency. A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasmids; excision repair-deficient rad1 and rad3 strains had repair efficiencies intermediate between those of rad52 and RAD cells. The level of repair also depended on the conditions of transformant selection; repair was more efficient in medium lacking tryptophan than in medium from which either histidine or uracil was omitted. The plasmid repair differential between these selective media was greatest in rad1 cells, and depended on RAD52. Plasmid-chromosome recombination was stimulated by psoralen damage, and required RAD52 function. Chromosome to plasmid gene conversion was seen most frequently at the HIS3 locus. In RAD and rad3 cells, the majority of the conversions were associated with plasmid integration, while in rad1 cells most were non-crossover events. Plasmid to chromosome gene conversion was observed most frequently at the TRP1 locus, and was accompanied by plasmid loss.

DNA↗
PubMed1994

Transformation of mycobacterial species using hygromycin resistance as selectable marker.

Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10(3)-10(5) transformants (micrograms DNA)-1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.

Bacterial Proteins↗
PubMed1988

Chicken homolog of the mos proto-oncogene.

We compared the sequence and properties of the chicken mos homolog with the previously characterized mouse and human c-mos genes. Sequence analysis revealed one major open reading frame of 1,047 base pairs encoding a protein of 349 amino acids. Both the nucleotide sequence and the deduced amino acid sequence showed 62% overall homology to mouse and human c-mos, but regions of higher conservation (approximately 70%) occurred in the putative ATP-binding and kinase domains. We detected mos transcripts by Northern (RNA) analyses in RNA prepared from chicken and quail ovaries and testes. Evidence for low levels of mos RNA expression in adult chicken heart, kidney, and spleen and in the entire embryo was obtained by S1 nuclease protection experiments. In contrast to the low transforming efficiency of human c-mos when linked to a mouse retroviral long terminal repeat element, chicken c-mos transformed NIH 3T3 cells as efficiently as mouse c-mos did. We also show that chicken primary embryo fibroblasts were morphologically altered when infected with an avian retroviral vector containing the chicken c-mos coding region.

Amino Acid Sequence↗