Highest Transformation Frequency Research Articles

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells.

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis.

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora.

High-frequency linear transformation elliptic filtersemploying minimum number of OTAs

High-frequency linear transformation (LT) elliptic filters employing a minimum number of operational transconductance amplifiers (OTAs) and capacitors are presented. Based on the LT, an OTA-C elliptic filter can be realised efficiently by adding capacitors to the relative all-pole filter as passive prototype counterparts. The canonical filter with all nodes having a desired capacitance, which is suitable for high-frequency operation, is developed. As an example, a new third-order elliptic lowpass filter is synthesised. Experimental results are obtained to verify the theoretical analysis. Furthermore, the proposed circuit can be extended to higher-order filters.

Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants

For the development of anAgrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing β-glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenic shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulentAgrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way.

Mantle cell lymphoma: Natural history defined in a serially biopsied population over a 20-year period

The histological, immunological and molecular characteristics of mantle cell lymphoma have only recently been delineated. Amongst these characteristics possible factors of prognostic significance include histological growth pattern and blastoid change. 66 previously untreated cases of mantle cell lymphoma were identified in a retrospective analysis. In 50 cases serial biopsies had been taken during the disease and in 20 cases autopsies had been performed. Besides established factors of prognostic significance, histological growth pattern and blastoid change were examined. 32 patients achieved an initial complete remission or good partial remission with most cases relapsing or progressing within 2 years. The median survival was 36 months. Factors predicting a poor outcome were high presenting stage, age > 70, low sodium, low albumin and splenomegaly. Blastoid transformation was also a poor prognostic feature, occurring in 32% of cases during life and in 70% of autopsies. Histological growth pattern had no influence on outcome. This study emphasises the difficulties in treating mantle cell lymphoma and the high frequency and prognostic importance of histological transformation.

Transformation of Potato (Solanum Tuberosum L.) using Tuber Discs and Stem Explants

ABSTRACTTransformation of three potato cultivars (Desiree, Isola and Anac) was achieved using tuber discs and callus tissue derived from cultured stems via co-cultivation with Agrobacterium tumafaciens Ti plasmid based vectors. Neomycin phosphotransferase (NPT II) marker and β-glucuronidase (GUS) reporter genes were used in transfer-optimization studies. First selection of transgenics was done on selective Murashige and Skoog (MS) media containing 100 mg/l kanamycin and 500 mg/l cefotaxime. Histochemical assays were done for GUS activity using X-Gluc substrat. In addition polymerase chain reaction based Southern blot analyses revealed the existence of the transferred genes. The highest transformation frequency (10%) was obtained with cultivar Desiree in MS media supplied with 5 mg/l zeatin riboside and 1.5 mg/l indolacetic acid. However, Anac gave the lowest frequency using tuber discs (3%) and stem explants (2%). This protocol could be useful for the improvement of potato through gene manipulation.

Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells.

In order to improve the in vitro transformation assay using BALB/3T3 cells, which is routinely conducted with minimal essential medium (MEM) containing 10% fetal calf serum (FCS), we examined the effect of a new medium after the cells had been treated with carcinogens. Preliminary experiments suggested that the use of T medium (modified DME/F-12) supplemented with insulin, transferrin, ethanolamine and sodium selenite plus 2% FCS resulted in a high transformation frequency. The present study confirmed that this medium was very efficient at inducing transformation foci. Transformation frequency was highest when 12-O-tetradecanoylphorbol-13-acetate (TPA) was started from 1 week after the carcinogen treatment. According to the new protocol using this medium, the transformation frequency was five times higher and transformation foci appeared much earlier than with the protocol using usual MEM plus 10% FCS medium. In addition, we tested several typical carcinogens and a promoter in order to confirm the applicability of the new protocol to the two-stage transformation assay. N-Methyl-N'-nitro-N-nitro-soguanidine (MNNG) and benzo[a]pyrene, but not noncarcinogenic benzo[e]pyrene, induced transformation foci. In the metabolic activation system dimethylnitrosamine gave a highly positive transformation. Okadaic acid, a non-TPA-type tumor promoter, enhanced transformation of MNNG-initiated cells. These studies demonstrate that the use of the proposed medium drastically improves transformation frequency in the two-stage in vitro transformation assay.

Chapter 3: Molecular Tools for Genetic Dissection of the Protozoan Parasite Toxoplasma gondii

Publisher Summary The genetic structure of Toxoplasma gondii is notable chiefly for being relatively conventional— similar to that of its mammalian host cells with respect to gene organization, codon usage, and nucleotide bias. These observations have led several investigators to examine the feasibility of molecular transformation in this parasite. This chapter outlines the use of several of the molecular genetic tools that have recently been developed for the T. gondii system. An introduction to parasite culture techniques is also provided. Recombinant molecules can be expressed either transiently or as stable transformants, as episomes or integrated into the parasite genome, and as single copy or multicopy transgenes. Stable integration can be produced by random nonhomologous recombination, single-site homologous recombination, or perfect gene replacement. Many of these outcomes can be selected specifically using appropriate vectors and transformation conditions. The extraordinarily high frequencies of stable transformation observed permit cloning by complementation, insertional mutagenesis/marker rescue, gene knock-outs, and allelic replacement. In combination with available classical and “cell-genetic” possibilities and physical and genetic mapping strategies, these tools provide a powerful arsenal for investigations into the biology of intracellular parasitism.

High frequency transformation of Alcaligenes eutrophus producing poly-?-hydroxybutyric acid by electroporation

A strain of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×102/μg DNA when 50 μl competent cells at 1010/ml were pulsed by 11.5 kV/cm for 5 ms with 1 μg DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25μF, a transformation efficiency as high as 1.4×107 transformants per μg of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli

A high frequency transformation system has been developed for Penicillium paxilli using pAN7-1. Up to 44% of the primary transformants were heterokaryons. Loss of hygromycin resistance was observed in primary transformants that were sub-cultured on non-selective media, but single spores of these primary transformants were mitotically stable on both selective and non-selective media. A molecular analysis of the transformants generated showed that 78% had single-site integrations, with half of these containing a single copy of pAN7-1. CHEF-gel electrophoresis showed that P. paxilli has at least six chromosomes with a total genome size of about 23.4 Mb.

High-frequency transformation from cultured cotyledons of Arabidopsis thaliana ecotypes ?C24? and ?Landsberg erecta?

Procedures were developed for rapid and prolific adventitious shoot regeneration of Arabidopsis thaliana (L.) Heynh ecotypes “Landsberg erect” and “C24” from cotyledon explants at 90–100% efficiency. Immature cotyledons had the highest shoot regeneration efficiency. Prolific regeneration was achieved in Murashige and Skoog medium (MS) supplemented with 0.1–0.4 mg−1 naphthalene acetic acid (NAA) and 1.0 mg−1 6-benzylaminopurine (BAP) within 2 weeks. The regenerated plants had a normal phenotype and produced fertile flowers and set seeds. The above regeneration protocol was used to develop a transformation method using disarmed strains of Agrobacterium tumefaciens strains pGV3850::pH1121 based on kanamycin (Km) selection. Transgenic shoots were produced within 2–3 weeks after inoculation. Transformation of shoots was confirmed by GUS histochemical assay, as well as southern blot hybridization.

High frequency vector-mediated transformation and gene replacement in Tetrahymena.

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.

Stable transformation of lettuce cultivar South Bay from cotyledon explants

Transgenic plants of lettuce cultivar (cv.) ‘South Bay’ were produced by using Agrobacterium tumefaciens vectors containing the β-glucuronidase (GUS) reporter gene and the NPT II gene for kanamycin resistance as a selectable marker. High frequency of transformation, based on kanamycin resistance and assays for GUS expression, was obtained with 24 to 72-h-old cotyledon explants cocultivated for 48 h with Agrobacterium tumefaciens. After the cocultivation period, the explants were placed in selection medium containing 50 or 100 mg l−1 of kanamycin, 100 mg l−1 cefotaxime and 500 mg l−1 carbenicillin for 10 days. Surviving explants were transferred every 14 days on shoot elongation medium. Progenies of R0 plants demonstrated linked monogenic segregation for kanamycin resistance and GUS activity.

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization.

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.

Factors affecting competence in a high frequency of transformation marine Vibrio

Summary: Natural plasmid transformation may be a mechanism for the horizontal transfer of non-conjugative plasmids in the marine environment, yet there are few marine model systems available for the study of this process. Using multimers of IncQ/P4 plasmids and a filter transformation assay, we have measured the effects of nutrients, salinity, temperature, as well as the development and maintenance of competence for genetic transformation in the high frequency of transformation (HFT) marine Vibrio strain WJT-1C. Transformation frequency was proportional to the amount of DNA used from 0.1 to 1.0 μg DNA and was saturated at concentrations greater than 1.0 μg. Competence began in the early-exponential phase and reached a maximum at the onset of stationary phase. Once attained, competence was maintained in both spent and nutrient-free media for at least 10 d. Thus, the establishment and maintenance of competence was unique compared to previously described transformation systems. Temperatures ranging from 4 to 33 °C had no significant effect on the maximal transformation frequency of WJT-1C, but at 37 °C the transformation frequency was reduced. However, temperature did affect the rate of the transformation process. Salinities in the range 12 to 50% had no significant effect on the transformation frequency but transformation frequencies were lower at 6% and 63%. Cells were transformed equally well in nutrient-free media or rich media. The ability of this marine HFT Vibrio strain to develop competence under a wide spectrum of conditions and to maintain the competent state indicates that natural plasmid transformation could occur in conditions found in tropical and subtropical estuaries.

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.