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PubMed1992

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency.

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5' non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5' regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.

Amino Acid Sequence↗
PubMed1988

Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker.

Recombinant plasmids have been obtained that lead to the accumulation of five- to ten-fold more puromycin-N-acetyl-transferase (PAC) mRNA and two- to three-fold more PAC activity than the already described plasmid pSV2pac [Vara et al., Nucl. Acids Res. 14 (1986) 4117-4124]. When these optimized recombinants were used for stable transformation to puromycin resistance, efficiencies up to 1 x 10(-2) were obtained, indicating that these pac-containing recombinants may be very useful dominant selectable markers for gene transfer in mammalian cells.

Acetyltransferases↗
PubMed1991

mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities.

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.

Animals↗
PubMed1984

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.

Adenoviruses, Human↗
PubMed1997

Effects of DNA topology on transformation efficiency of Bacillus subtilis ISW1214 by electroporation.

We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonculease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6 +/- 0.6) x 10(5) transformants per microgram DNA higher than that (2.0 +/- 0.3) x 10(4) transformants per microgram DNA for the cr DNA, using the DNA concentration of 20 micrograms/ml at an electric field strength of 7 kV/cm and a capacitance of 10 microF with a single decayed pulse. The transformation efficiency (TE) for the ln DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.

Animals↗
PubMed1995

Protoplast fusion method for efficient transformation of mouse spermatogenic cells.

The efficiencies of methods for introducing DNAs into spermatogenic cells were investigated. The calcium phosphate method has been most commonly used to transfect DNA into cultured cells, but this method severely damages spermatogenic cells. Therefore, we investigated the efficiencies of transfection of spermatogenic cells by DEAE-dextran, modified liposome, and protoplast fusion methods, which seemed to cause less damage to spermatogenic cells than the calcium phosphate method. The protoplast fusion method was the most suitable for transfection of spermatogenic cells.

Animals↗
PubMed2001

[The factors of improving rice transformation efficiency].

The factors influencing the rice transformation frequency have been investigated by using 8 indica and japonica rice cultivars during the transformation procedures, such as induction of calli, selection, and regeneration. The following is the results: The immature embryos were pretreated in 4 degrees C for 4 or 7 days before inoculation, and the regeneration frequency of calli would be increased. High osmotic treatment may increase the possibility of transformation, especially for calli of 6 months or older. The transformation frequency was improved when selection pressure was removed during differentiation period. The V-type selection may be better than the normal selection. There is a difference of differentiation ability among the calli of three periods and it is found that the calli of two or three months are the best for transformation. Proline and DMSO can decrease browning rate of calli.

Dimethyl Sulfoxide↗
PubMed1991

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.

Ascomycota↗
PubMed1988

High-efficiency transformation of bacterial cells by electroporation.

We have developed a method for efficiently generating transient pores in the outer membranes of Escherichia coli K-12 derivatives by using a new type of electroporation apparatus. The pores are large enough and persist long enough to facilitate the equilibration of plasmid molecules between the intracellular and extracellular spaces. The method has been used to transform bacterial cells with an efficiency greater than 10(9) transformants per microgram of plasmid. It has also been used to extract intact plasmid from transformed cells with efficiencies comparable to those of the traditional alkaline lysis or CsCl equilibrium density gradient techniques. The technique is simple and rapid, allowing a transformation or the preparation of microgram quantities of plasmid to be accomplished in minutes.

Cell Membrane Permeability↗
PubMed1969

"Rescued" SV40: increased transforming efficiency in mouse and human cells.

Plaque-size mutants of simian virus 40 (SV40) have been characterized with respect to their ability to transform mouse and human cells in vitro. Large plaque mutants of SV40 were extremely inefficient at transforming mouse or human cells, requiring approximately 50 times more infectious particles per transformation event than did the small plaque mutants. The minute plaque SV40 was the most efficient, requiring less than 10(3) plaque-forming units per transformation in mouse Balb/3T3 cells and only 1.5 x 10(4) plaque-forming units per transformation unit in the "susceptible" human strains. Both small and minute plaque virus were unable to help the large plaque virus to transform. Virus "rescued" from transformed 3T3 cells by cocultivation with monkey kidney cells was more efficient at transformation than the parental type. The increased efficiency could be shown for each plaque type and in human as well as in mouse cells. The property of "high efficiency transformation" is stable through at least two passages in monkey kidney cells.

Animals↗
PubMed1980

Biological activity of cloned Moloney sarcoma virus DNA: Terminally redundant sequences may enhance transformation efficiency.

We have measured the ability of cloned restriction fragments containing the whole and partial genomes of two strains of Moloney murine sarcoma virus to induce cell transformation in DNA transfection assays. The cloned intact ml and HTl murine sarcoma virus proviruses transform with an efficiency of approximately 40,000-50,000 focus-forming units/pmol of proviral DNA, and the majority of these transformed cells contain a rescuable viral genome. A cloned 2.1-kilobase-pair internal fragment of the murine sarcoma virus containing 1.2 kilobase pairs of sarcoma virus-specific sequences (src) and approximately 900 base pairs of leukemia virus-derived sequences adjacent to the 5' end of src transforms with approximately 1/10,000th the efficiency of the intact genome. When leukemia virus-deprived sequences containing a single copy of the 600-base-pair direct terminal repeated sequences are present at either the 5' or 3' end of this src-containing fragment, the transforming activity is stimulated 1000-fold. Cotransfection with a mixture of cloned fragments, one containing the internal 2.1-kilobase-pair src fragment and the other containing a single copy of the terminally redundant sequence, results in a 300-fold increase in transformation efficiency.

Animals↗
PubMed1991

Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene.

An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.

Amino Acid Sequence↗
PubMed1994

Differential transformation efficiency but not AP-1 induction under anchorage-dependent and -independent conditions.

The JB6 mouse epidermal cell system has been extensively used as an in vitro model for the study of tumor promotion. The present study aimed to assess the relevance of monolayer measurements to the process of transformation, which is induced more efficiently under anchorage-independent (AI) conditions. Although it would be ideal to use identical conditions for studying tumor promoter-induced transformation and biochemical and molecular events that may cause the process, it is not feasible in the case of soft agar conditions because cells cannot be readily recovered. In the present report, we used liquid medium over agar as an AI condition that permitted efficient recovery of cells. Responses to tumor promoter have been compared with those in monolayer and semisolid agar. Results indicate that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced similar magnitude concentration-dependent transformation of JB6 cells under both of the AI conditions, namely soft agar and over-agar. Under anchorage-dependent (AD) conditions of exposure to TPA, the transformation efficiency was much lower than that seen under AI conditions. Mechanical detachment of monolayer cells after 5-10 days TPA exposure enriched the transformed phenotype. Activator protein 1 transcriptional activity measured at 12 h was induced equally under AD and AI conditions, and thus is not an early limiting event that could explain the lower transformation efficiency seen under AD conditions. To summarize, the over-agar and monolayer assays described in this study can be considered valid for the study of early biochemical and molecular events relevant to the promotion of transformation measured in soft agar.

Animals↗
PubMed1998

High-efficiency transformation of Chlamydomonas reinhardtii by electroporation.

We have established a high-efficiency method for transforming the unicellular, green alga Chlamydomonas reinhardtii by electroporation. Electroporation of strains CC3395 and CC425, cell wall-less mutants devoid of argininosuccinate lyase (encoded by ARG7), in the presence of the plasmid pJD67 (which contains ARG7) was used to optimize conditions for the introduction of exogenous DNA. The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 x 10(5) transformants per microg of DNA; this frequency is two orders of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA. The electroporation procedure described in this article is of general utility, and makes it feasible to isolate genes by direct complementation of Chlamydomonas reinhardtii mutants.

Animals↗
PubMed1999

A novel method for increasing the transformation efficiency of Escherichia coli-application forbacterial artificial chromosome library construction.

Bacterial artificial chromosome (BAC) libraries play a pivotal role in genomics studies. A crucial step in BAC library construction is the transformation of Escherichia coli by electroporation. Absolute efficiency (cfu/microgram DNA) is affected by a number of factors including the topological form and treatment of DNA samples. Here we report a simple new protocol using tRNA assisted precipitation that increased transformation efficiency by 70-fold for BAC ligations and up to 400-fold for plasmid ligations. The mechanism may involve altering or stabilizing the topographical form of the DNA molecules.

Chromosomes, Bacterial↗
PubMed2025

Systematic Optimization Enables Near-Perfect In Vitro Transformation Efficiencies for Spirodela polyrhiza (Greater Duckweed).

The in vitro transformation of plants, or the delivery of foreign genetic material that is incorporated into their genomes, represents a powerful tool both for elucidating genotype-phenotype relationships and for generating plant cultivars which have desirable traits for agriculture and/or biotechnological applications. However, outside of a few model species, the processes involved in transformation are often inefficient and can take months to perform for many plant species, with several bottlenecks occurring at the different stages of calli induction, genetic transfection, and plant regeneration. While duckweeds - aquatic monocots whose species include some of the smallest and fastest-growing flowering plants on the planet - have distinguished themselves with several emerging biotechnological applications, they too are the subject of conflicting reports regarding their transformation potential and ability to be genetically manipulated. Here, we synthesized and optimized the protocols for in vitro transformation of duckweed Spirodela polyrhiza (Greater Duckweed) from start-to-finish: achieving >90% - 100% efficiencies for each of calli induction; transient and stable genetic transformation; visual marker-free selection of transformants; and regeneration of genetically modified plants with stable transgene expression for over 100 generations - and which in S. polyrhiza can be achieved over the course of weeks instead of months. The integrated, streamlined approaches for all stages of in vitro transformation overcome many bottlenecks and can help to pave the way for high-throughput functional genomics studies and synthetic biology applications in this biotechnologically-important species.

CRISPR/Cas9↗
PubMed1991

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15-20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4-5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and beta-glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7-8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.

Cells, Cultured↗
PubMedDate not supplied

Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations.

A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p beta-Gal plasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium+ ++) bromides random copolymer (C16PVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma "Jurkat", and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The in vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of beta-galactosidase (beta-Gal) expression using 4-methylumbeliferril beta-D-galactopyranoside as a substrate (p beta-Gal plasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗