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 829 records · Page 46Linked to original sources
PubMed2000

[Establishment of a method for GUS gene transferring into wheat (Triticum astivum L.) embryos by low energy ion beam implantation].

Physical parameters influencing transformation of wheat mediated by low energy ion beam, including type of ion, parameters of ion energy, dose and dose rate, were studied. Ar+ was regarded as suitable ions implanted in transformation. 20-25 keV of energy, 4.68 x 10(16) ions/cm2 of dose, 2.6 x 10(15) ions/cm2 of dose rate were chosen as appropriate implantation parameters. The suitable culture conditions for induction and growth of callus and the optimal selection scheme were established, After implantation and selection, resistant calli and hygromycin-resistant plantlets were obtained from three varieties. Molecular analysis data proved that GUS gene had integrated into the wheat genome. The mature embryo transformation efficiency of wherat variety Yangmai 5, Yangmai 158, Wanmai 32 reached 9.5%, 10.8%, 11.2% measured in produced hygromycin-resistant callus and 1.4%, 3.4%, 1.7% measured in regenerated plants, respectively, This experiment provides a basis for further investigation of wheat transformation system. Low energy ion beam mediated transformation can be extended to other plant recalcitrant to Agrobacterium tumefaciens as soon as methodological parameters are optimized.

Gene Transfer Techniques↗
PubMed2006

[Transformation of embryogenic Calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) mediated by agrobacterium].

Formation of embryogenic calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) was induced from mature seeds as explants, and proliferated on MS medium containing 2,4-D 5.0 mg/L and KT 0.05 mg/L. An effective and stable callus regeneration system was established by optimizing the culture conditions (Tables 1, 2 and Fig.2). After the calli were subcultured 8 weeks, selected the whitish-yellow-coloured compact nodular calli that transformed with plasmid pCAMBIA1304 carrying hygromycin resistance gene (hptII) and Pseudomonas pseudoalcaligenes insecticidal protein gene (ppIP), which was mediated by an Agrobacterium strain EHA105. Resistant plants were obtained after hygromycin selection (Figs.3, 4). Some important factors that affect the transformation efficiency were studied, which included selection pressure, time of embryogenic calli proliferation, OD value of Agrobacterium suspension, temperature, medium and time of co-cultivation, and concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformation plant regeneration. This research is the first successful genetic transformation of Elymus sibiricus L. cv. Chuancao No.2 mediated by Agrobacterium.

Culture Media↗
PubMedDate not supplied

Electrotransformation of Yersinia ruckeri by plasmid DNA.

Yersinia ruckeri, a fish pathogenic bacterium in aquaculture, was used to evaluate the electroporation as a new transformation method for this species. DNA used for the electrotransformation were plasmids of molecular mass ranging from 2.3 kb to 33 kb, and diverse replicons. To optimize this method we used Y. ruckeri 11.29 strain (from serotype 02) and pSU2718 DNA. The best transformation efficiency (6.0 x 10(5) transformants/micrograms DNA) was obtained with 12.5 kV/cm, 25 microF, 400 omega and 2 hours of incubation after pulse. When these conditions were applied to other strains belonging to different serotypes and other plasmids, we obtained transformants in all strains assayed, but only when using low molecular weight plasmids. Plasmid vectors and resident plasmid were not modified in host strains after electrotransformation. In studies of conformation we confirmed that only circular DNA was able for transformation. The utilization of this technique for direct cloning in Y. ruckeri makes possible further studies on recombinant DNA.

Cell Division↗
PubMed1993

[The C-Jun oncoprotein].

Jun and Fos are major components of the transcriptional complex AP-1 (Activator Protein-1), a collection of dimeric transcriptional activators composed of members of the Jun and Fos family of bZIP proteins, that bind to a common site known as TRE (TPA Responsive Element) or the AP-1 site. Transcription of c-jun is rapidly induced by exposure to different extra-cellular signals like growth factors, cytokines, tumor promoters (TPA), UV and other DNA-damaging agents. Transcriptional activation of c-jun is a two step mechanism. First, the pre-existing c-Jun protein is activated by posttranscriptional modifications, and second, modified c-Jun activates its own transcription, and the expression of AP-1-dependent genes. Modifications of c-Jun include dephosphorylations, phosphorylations and oxydo-reduction. The transcriptional activation by c-Jun is modulated by heterodimerization with other members of the bZIP family of proteins, and by transcriptional interference with other transcription factors like some members of the hormone nuclear receptors, or MyoD. AP-1 is tightly associated to both the control of cell proliferation and the oncogenic process. Constitutive activation of AP-1 leads to cell transformation in vitro, probably due to the accumulation of homodimeric c-Jun:c-Jun complexes. This hypothesis has been directly confirmed by constructing c-Jun hybrid proteins capable to form only homodimers. Deregulated expression of such proteins efficiently transforms primary cells in culture. These hybrid proteins constitute a powerful tool in order to identify new cellular functions AP-1-dependent, involved in the control of cell proliferation.

Alpharetrovirus↗
PubMed1976

Host range temperature-sensitive mutants of herpes simplex virus type 2.

Two small-plaque mutants of herpes simplex virus type 2 (HSV-2) (strain 333), whose growth at 39 C was blocked in certain cell types (cell-dependent temperature sensitivity), were compared compared with parental virus in a number of biological assays. One mutant (no. 69) was found to produce a large number of morphologically normal, but noninfectious, particles; under nonpermissive conditions, these mutant particles were able to interfere with the replication of wild-type HSV-2. The other mutant (no. 74), which is known to belong to a different complementation group, appeared to direct little virus DNA synthesis, even at the permissive temperature. Progeny production and virus DNA synthesis in cells infected by mutant 74 were delayed in comparison with wild-type virus-infected cells. Both mutants were found to be more sensitive to UV irradiation than the parental virus; this was especially marked in the case of mutant 74. Moreover, this mutant was found to have a high transforming efficiency at much lower doses of irradiation than those needed to abolish the cytopathic effect of wildtype HSV-2.

Animals↗
PubMed2002

Expression of an antisense GIGANTEA (GI) gene fragment in transgenic radish causes delayed bolting and flowering.

A late-flowering transgenic radish has been produced by the expression of an antisense GIGANTEA (GI) gene fragment using a floral-dip method. Twenty-five plants were dipped into a suspension of Agrobacterium carrying a 2.5 kb antisense GI gene fragment from Arabidopsis, along with the gusA and bar reporter genes, all under the control of a CaMV 35S promoter. From a total of 1462 seeds harvested from these floral-dipped plants, 16 Basta-resistant T1 plants were found to have GUS activity (transformation efficiency of 1.1%). Southern analysis confirmed the integration of one or two copies of the gusA gene in these herbicide-resistant plants. Expression of the GI gene in T1 plants was much reduced compared to both wildtype plants and plants transformed with pCAMBIA3301 (positive control). In the progenies of eleven T1 plants analysed (T2 generation), all lines showed a significant delay in both bolting and flowering times compared to wildtype and positive control plants, and that, the level of GI transcript was inversely proportional to the time of bolting and flowering. At a maximum, bolting and flowering times were delayed by 17 and 18 days respectively, compared to wildtype plants (in positive control plants, the delay was 23 and 26 days, respectively). Ten of the 11 lines exhibited a significant reduction in plant height compared to wildtype and positive control plants. This study provides evidence that down-regulation of the GI gene by co-suppression could delay bolting in a cold-sensitive long-day (LD) plant. Production of late-flowering germplasms of radish may allow this important crop to be cultivated over an extended period and also provide further food to the famine countries of S/E Asia.

Agrobacterium tumefaciens↗
PubMed1992

Gene transfer to tumor-infiltrating lymphocytes and other mammalian somatic cells by microprojectile bombardment.

We demonstrate the application of particle delivery to the transformation of mammalian somatic cells. Two mouse T-lymphocyte cell lines and Chinese hamster ovary (CHO) cells were transformed transiently with the RSV-ADH (alcohol dehydrogenase) gene. Stable gene transfer was also demonstrated in CHO cells at a frequency of 6 x 10(-4) and in T-cells at a frequency of 1 x 10(-4), using beta-Gal/neomycin-resistance gene constructs. The helium gas acceleration mechanism (PDS 1000/He) and particle delivery method allowed better velocity control and particle dispersion than the gunpowder driven instrument. These improvements have increased cell viability, transformation efficiency, and cellular targets.

Air Pressure↗
PubMed1986

Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions.

We examined bovine papillomavirus type 1 (BPV-1) DNAs mutated in the E2 open reading frame (ORF) to determine their ability (i) to transform C127 cells and (ii) to remain extrachromosomal in transfected cells. Results obtained with deletion mutants and insertion mutants containing a linker with translational termination codons in all possible reading frames indicated that an E2 ORF gene product(s) is necessary for efficient transformation, as well as viral plasmid replication and maintenance in the context of the full BPV-1 genome. Complementation assays in which mutant BPV-1 DNAs were transfected into cell lines expressing some viral functions from integrated BPV-1 cDNAs demonstrated that the E2 ORF product, when provided in trans, could allow BPV-1 E2 mutants to remain extrachromosomal. The E2 function could also augment transformation of some, but not all, BPV-1 E2 mutants, allowing identification of another region of BPV-1 involved in cellular transformation. It is likely that the role of the BPV-1 E2 product(s) in transformation and plasmid maintenance is indirect. A BPV-1 mutant altered in the E5 ORF is transformation defective and unable to replicate as a stable plasmid in C127 cells.

Animals↗
PubMed1997

A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.

Amino Acid Sequence↗
PubMed2002

Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic differentiation.

Tbx3 is a member of the T-box family of transcription factors. Mutations in Tbx3 cause ulnar-mammary syndrome, an autosomal dominant disorder characterized by upper limb defects, apocrine-gland defects including mammary hypoplasia, and tooth, hair and genital defects. In cell culture, Tbx3 and its close relative Tbx2 are capable of immortalizing mouse embryo fibroblasts. We show that expression of Tbx3 together with Myc or oncogenic Ras (H-Ras(Val17)) leads to efficient transformation of mouse embryo fibroblasts. Oncogene cooperation by Tbx3 correlates with an ability of Tbx3 to suppress the induction of p19ARF and p53 that is typically caused by overexpression Myc and Ras, and to protect against Myc-induced apoptosis. Whereas Tbx3 is capable of interfering with apoptosis caused by excessive Myc levels, a Tbx3 mutant lacking its C-terminal repression domain shows no anti-apoptotic activity and fails to repress levels of p19ARF or p53. Consistent with an ability to suppress p53 pathway function, we find that Tbx3, but not a Tbx3 C-terminal mutant, efficiently blocks myogenic differentiation of C2C12 myoblasts. Our results support the idea that deregulation and/or excessive levels of Tbx3 may have oncogenic potential in vivo.

3T3 Cells↗
PubMed2006

Development of genotype-independent regeneration system for transformation of rice (Oryza sativa ssp. indica).

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L(-1) thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5-8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L(-1) carbenicillin or 250 mg L(-1) cefotaxime to kill the rice shoot apical meristem was 50 mg L(-1) and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T(0) transformant plantlets.

Acetophenones↗
PubMed1984

Transformation by purified early genes of simian virus 40.

A rapid soft-agar assay using baby hamster kidney (BHK21 cl.13) cells has been developed to establish the functional roles for the large T and small t antigens of SV40 in transformation. Plasmids expressing either large T or small t antigens of SV40 have also been constructed and these plasmids have been used separately or in combination for transformation. A large T clone, pD3-05, containing a deletion in the small t-specific coding region [0.584-0.54 map units (mu)], transformed a low-background subclone of baby hamster kidney (BHK21 cl.13) cell line and F111 rat fibroblasts to anchorage independence at a low level (10-20 and 1%, respectively, of an early region clone from wild type [WT], pW2). A WT-derived small t clone, pW2-t, containing a deletion in the large T-specific coding region (0.373-0.169 mu), did not transform F111 cells, but transformed BHK21 cells at a very low level (about 2% of pW2). Another WT-derived small t clone, pW2-t/B1, containing a larger deletion in the large T-specific coding region (0.512-0.169 mu), did not transform either BHK21 or F111 cells. However, cotransformation with pD3-05 clone and pW2-t or pW2-t/B1 clone increased the frequency of transformation to about the same level as that of pW2. The ability of the small t clones to enhance the transformation efficiency of the large T clone was not due to recombination between the two plasmids, since cotransformation with pD3-05 and a small t clone without the polyadenylation [poly(A)] signal sequence from WT, pW-t8, did not increase the frequency of transformation. When the frequency of transformation was determined by the focus assay using F111 cells, pD3-05 transformed as well as pW2. Also, cotransformation with pD3-05 and pW2-t/B1 did not increase the frequency of focus formation. Therefore, the small t antigen was not required for this morphological transformation.

Animals↗
PubMed1980

Biochemical transformation by temperature-sensitive mutants of herpes simplex virus type 1.

Biochemical transformation assays of herpes simplex virus type 1 temperature-sensitive (ts) mutants distinguished three groups of mutants with regard to their thymidine kinase (TK) transforming ability: those incapable of transferring the TK gene at either the permissive or restrictive temperatures (group I); those resembling the wild-type virus, and therefore able to transform at both the permissive and nonpermissive temperatures (group II); and those that failed to transform or exhibited very low transformation frequencies at the permissive temperature but were able to transform at the nonpermissive temperature (group III). Two mutants in group II exhibited greatly enhanced transformation efficiency at the permissive temperature. The ts lesions in the majority of the mutants tested map between 0.30 and 0.60 units on the viral genome. Mutants with TK-positive (TK+), but DNA-negative, phenotypes at the nonpermissive temperature produced no TK+ transformants at the permissive temperature and only unstable transformants at the nonpermissive temperature. This suggests that a function which is required for viral DNA synthesis is also required to obtain stable expression or to transfer the TK+ gene or both when transfer is mediated by the entire viral genome.

Cell Line↗
PubMed1997

Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae.

Two delta-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first vector was a traditional construct containing only one delta sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low. The second carried two delta sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double delta vector was employed, the integrated copy number was more than doubled relative to the single delta system and final beta-galactosidase levels exceeded those obtained with the 2 mu-based plasmid. Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single delta vector.

Genetic Vectors↗
PubMed2006

Enhancement of biomolecule transport by electroporation: a review of theory and practical application to transformation of Corynebacterium glutamicum.

Selective and reversible permeabilization of the cell wall permeability barrier is the focus for many biotechnological applications. In this article, the basic principles for reversible membrane permeabilization, based on biological, chemical, and physical methods are reviewed. Emphasis is given to electroporation (electropermeabilization) which tends to be the most popular method for membrane permeabilization and for introduction of foreign molecules into the cells. The applications of this method in industrial processes as well as the critical factors and parameters which affect the success of this approach are discussed. The different strategies developed throughout the years for increased transformation efficiencies of the industrially important amino acid-overproducing bacterium Corynebacterium glutamicum, are also summarized.

Biological Transport↗
PubMed2001

A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium.

Fleshy fruits represent a very important economic resource and, therefore, they are an ideal target for biotechnological ameliorations. However, because of their physiological and anatomical characteristics, ripe fleshy fruits represent an extremely difficult material for transient gene expression assays aimed at the study of gene promoters in a short time. To this purpose, a fast and efficient Agrobacterium-mediated transient gene expression system was developed for ripe fleshy fruits. A beta-glucuronidase reporter gene interrupted by an intron was used in order to prevent the possible expression of GUS activity by the Agrobacterium cells. The contemporary use of another reporter gene was used to check the transformation efficiency. This method is based on the injection of an Agrobacterium suspension into the fruits, and allows both qualitative and quantitative assays in a wide range of fruits to be carried out.

Fruit↗
PubMed1987

Functional relatedness between the E1a and E1b regions of group C and group D human adenoviruses.

The functional relatedness of the transforming genes (E1a and E1b) of adenovirus type 9 (group D) which induces mammary tumors in rats and those of the non-tumorigenic adenoviruses, Ad2 and Ad5 (group C) was examined. Transfection of established rat embryo cells with a DNA segment containing the E1a and E1b regions of Ad9 resulted in efficient transformation; similar results have been shown for group A, B and C Ads. In contrast to Ads of group A, B and C, Ad9 DNA containing the E1 region or the entire viral genome was unable to transform primary baby rat kidney (BRK) cells. The functional relatedness of genes encoded within the E1 region was compared using a mutant complementation assay in which various group C mutants defective in the entire E1 region or in the E1a or E1b regions alone as well as mutants defective exclusively within the 19K or 58K T antigens coding regions of E1b were coinfected with wild type (wt) Ad9 and tested for group C mutant DNA replication, virus production, or expression of early and late genes. These studies have shown that a defect in the entire E1 region of Ad2 could only be complemented poorly by Ad9; our earlier studies have shown that coinfection with Ad12 (group A) or Ad7 (group B) resulted in efficient complementation (Brusca and Chinnadurai (1981) J. Virol. 39, 300-305). Further analysis indicated that a defect in the E1a region could be complemented by the group D E1a region. The level of E1a complementation as judged by mutant DNA replication and activation of expression of mutant early viral genes was about one-fourth to one-fifth the level in 293 cells that constitutively express Ad5 E1a and E1b regions. Our results indicate that a defect in the E1b 19K T antigen, which leads to degradation of intracellular DNA in infected cells, could be complemented by the group D protein. However, a defect in the E1b 58K T antigen could not be efficiently complemented by the group D protein. Coinfection of group C mutants defective in the 58K T antigen and Ad9 wt did not lead to an increase in the mutant viral production. Furthermore, in cells coinfected with the 58K T antigen mutants and Ad9 wt there was a large reduction in the accumulation of group C late cytoplasmic RNA. The observed complementation defect of Ad9 in supporting multiplication of group C mutants defective in the entire E1 region may therefore be a cumulative effect of both E1a and E1b regions.

Adenovirus Early Proteins↗
PubMed1987

Repair of UV-irradiated plasmid DNA in Saccharomyces cerevisiae. Inability to complement mutational defects in excision repair by in vitro treatment with Micrococcus luteus UV endonuclease.

Excision repair defects of Saccharomyces cerevisiae rad1-1, rad4-4, rad7-1 and rad14 mutants were examined. As previously found, transformation of such cells with UV-irradiated plasmid DNA is poor compared to wild-type yeast. Treatment of UV-irradiated YRp12 plasmid DNA with crude preparations of Micrococcus luteus UV endonuclease before introducing it into rad1-1 cells increased transformation efficiency to wild-type levels. This is consistent with earlier reports of rad1-1 mutants being defective in the incision step of excision repair. However, with purified UV endonuclease little or no rescue occurred when the UV-irradiated plasmid was incised before transformation into rad1-1 or rad4-4 cells. Furthermore, the purified UV endonuclease reduced transformation of rad7-1 and rad14 mutants to levels seen in rad1-1 and rad4-4 cells. In contrast such treatment caused only a small decrease in the transforming ability of UV-irradiated DNA in wild-type cells. These results show that yeast can normally process pre-incised, UV-irradiated DNA and that this activity is absent in rad1-1, rad4-4, rad7-1 and rad14 mutants. Thus, in addition to their previously reported roles in incision, the RAD1, 4, 7 and 14 gene products are also required for repair to continue after the incision of DNA lesions.

DNA Repair↗