Lipofectin: direct gene transfer to higher plants using cationic liposomes

Summary It has recently been shown that lipofectin, a commercially available preparation of cationic liposomes is capable of animal and plant cell line transfection. Here, it is analyzed with respect to its toxicity for higher plant protoplasts and used for transient expression and stable transformation experiments with mesophyll protoplasts of Nicotiana tabacum and Nicotiana plumbaginifolia. Transient expression of the -glucuronidase gene (GUS) under control of the CaMV-35S-promoter was lower than after introduction of the same gene by polyethylene glycol. By transferring the neomycin phosphotransferase gene (NPTII) and subsequent culture and regeneration under selection with kanamycin, stably transformed plants were recovered after using Lipofectin in various protocols with or without additional application of electroporation. Efficiencies of stable transformation were comparable to those achieved with PEG and/or electroporation. Confirmation of transformants included assaying the enzyme activity of the gene product, genomic blotting, and transfer of the resistant phenotype to the progeny produced from selfed primary transformants.     

High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens

 Agrobacterium tumefaciens efficiently transforms most plants. A few dicotyledonous plants and most monocotyledonous plants are, however, recalcitrant to A. tumefaciens infection. We investigated whether the constitutive synthesis of a high level of the T-strand DNA intermediate can improve the transformation efficiency of plants. We previously described a mutation in the vir gene regulator virG, virGN54D, that allows constitutive expression of the vir genes. We also described the isolation of a mutant plasmid that is present at a significantly high level in A. tumefaciens. The two mutations were combined to produce an A. tumefaciens strain that synthesizes a high level of T-strand DNA in an inducer-independent manner. DNA transfer efficiency of the mutant was measured by monitoring β-glucuronidase (GUS) expression in a transient transfer assay. A significant increase in the efficiency of DNA transfer to both rice and soybean was observed with the double mutant. The presence of virGN54D had a major positive effect on transformation efficiency. Received: 4 August 2000 / Revision received: 9 October 2000 / Accepted: 12 October 2000     

A convenient set of vectors for expression of multiple gene combinations in plants

A set of vectors has been developed that simplify shuttling expression cassettes between small plasmids of high copy number ideal for experiments involving biolistic transient expression and a binary transformation plasmid. Three cassettes for the expression of a cloned coding sequence behind different promoters have been modified; combinations of these cassettes can be excised withNot I, and sequentially cloned into the transformation vector in a procedure that removes the first cloning site. The system is demonstrated by inducing anthocyanin synthesis with paired regulatory genes of maize biolistically delivered to a maize cell suspension, and then expressed in transformed tobacco.     

A probable lipid transfer protein gene is induced by NaCl in stems of tomato plants

A full-length tomato cDNA clone, TSW12, which is developmentally and environmentally regulated, has been isolated and characterized. TSW12 mRNA is accumulated during tomato seed germination and its level increases after NaCl treatment or heat shock. In mature plants, TSW12 mRNA is only detected upon treatment with NaCl, mannitol or ABA and its expression mainly occurs in stems. The nucleotide sequence of TSW12 includes an open reading frame coding for a basic protein of 114 amino acids; the first 23 amino acids exhibit the sequence characteristic of a signal peptide. The high similarity between the TSW12-deduced amino acid sequence and reported lipid transfer proteins suggests that TSW12 encodes a lipid transfer protein.     

Polyethylene glycol-mediated direct gene transfer in Nicotiana spp.

Direct gene transfer to protoplasts is a widespread experimental procedure. In this review, it is discussed in relation to the fate and expression of transforming DNA and its genetic transmission upon integration, and expression (in)stability during successive meiotic generations. The results suggest that the major differences between Nicotiana plumbaginifolia and N. tabacum were not in DNA uptake but rather in the specific fate of the transforming DNA. Differences between integrative transformation versus transient gene expression are discussed. Transient gene expression experiments with cotransformed independent constructs enable accurate assays of gene activity modulation, including both activation and repression. The study of the time of integration of foreign DNA using haploid mesophyll protoplasts showed that foreign DNA integration took place during the first two division cycles of the protoplasts. Most of the transfomants inherited the foreign genes as a monogenic trait: N. plumbaginifolia exhibited a higher proportion of multiple insertions compared with N. tabacum . Furthermore, gene integration occurs as a random process. Compared to 'simple' transformation, cotransformation with independent constructs showed a higher level of rearrangement of the inserted DNA. Foreign gene transmission, followed through different generations, indicated that inactivation of the inserted gene could happen through a variety of processes. These results open up a series of questions concerning the applied aspects of direct gene transfer to plants.     

Novel methods for adenovirus-mediated gene transfer to blood vessels in vivo

Adenovirus-mediated gene transfer is a promising method for studies of vascular biology and potentially for gene therapy. Intravascular approaches for gene transfer to blood vessels in vivo generally require interruption of blood flow and have several limitations. We have used two alternative approaches for gene transfer to blood vessels in vivo using perivascular application of vectors. First, replication-deficient adenovirus expressing nuclear-targeted bacterial b-galactosidase was injected into cerebrospinal fluid via the cisterna magna of rats. Leptomeningeal cells over the major arteries were efficiently transfected, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. When viral suspension was injected with the rat in a lateral position, the reporter gene was expressed extensively on the ipsilateral surface of the brain. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, positioning of the head may target specific regions of the brain. Second, vascular gene delivery was accomplished by perivascular injection of virus in peripheral vessels. Injection of the adenoviral vector within the periarterial sheath of monkeys resulted in gene transfer to the vessel wall that was substantial in magnitude although limited to cells in the adventitia. Approximately20% of adventitial cells expressed the transgene, with no gene transfer to cells in the intima or media. These approaches may provide alternative approaches for gene transfer to blood vessels, and may be useful for studies of vascular biology and perhaps vascular gene therapy.     

Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct gene transfer to protoplasts: Fate of the transferred genes

Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.     

Liposome-mediated gene transfer in fish embryos

Liposome-mediated gene transfer was used to introduce large DNA constructs into zygotes of African catfish. The technique is based on the delivery of recombinant bacteriophage lambda particles (or DNA-protamine complexes) into the cytoplasm of target cells by negatively charged, large unilamellar liposomes. Dechorionated zygotes and early fish embryos were treated with the transforming liposomes. Expression of the introduced reporter genes during the first three weeks of the development of the larvae was followed by measuring the activity of corresponding enzymes. These assays have indicated very efficient DNA uptake into the embryos.     

The Chlorella virus adenine methyltransferase gene promoter is a strong promoter in plants

An upstream region isolated from a eukaryotic algal virus adenine methyltransferase gene was tested for promoter function in plants. Fusion of this region to the chloramphenicol acetyltransferase reporter gene resulted in significantly higher expression than fusion with the cauliflower mosaic virus 35S promoter. Strong levels of expression were also found in electroporated monocot plant cells. The promoter activity in transgenic tobacco plants showed tissue-specific expression. Leaves had the highest expression followed by stems and flowers. The promoter activity was not detected in root tissue. Environmental cues, such as light, and the phytohormones auxin and cytokinines had no effect on the promoter's expression. This promoter might be utilized to achieve high levels of expression of introduced genes in higher plants.     

A likelihood framework to measure horizontal gene transfer

We suggest a likelihood-based approach to estimate an overall rate of horizontal gene transfer (HGT) in a simplified setting. To this end, we assume that the number of occurring HGT events within a given time interval follows a Poisson process. To obtain estimates for the rate of HGT, we simulate the distribution of tree topologies for different numbers of HGT events on a clocklike species tree. Using these simulated distributions, we estimate an HGT rate for a collection of gene trees representing a set of taxa. As an illustrative example, we use the "Clusters of Orthologous Groups of proteins" (COGs). We also perform a correction of the estimated rate taking into account the inaccuracies due to gene tree reconstructions. The results suggest a corrected HGT rate of about 0.36 per gene and unit time, in other words 11 HGT events have occurred on average among the 44 taxa of the COG species tree. A software package to estimate an HGT rate is available online (http://www.cibiv.at/software/hgt/).     

Adeno-associated virus-mediated gene transfer

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system.     

Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413

 The use of genetically engineered crop plants has raised concerns about the transfer of their engineered DNA to indigenous microbes in soil. We have evaluated possible horizontal gene transfer from transgenic plants by natural transformation to the soil bacterium Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA from two sources of transgenic plant DNA and different forms of plasmid DNA with an inserted kanamycin resistance gene, nptII, were measured. Clear effects of homology were seen on transformation frequencies, and no transformants were ever detected after using transgenic plant DNA. This implied a transformation frequency of less than 10^-13 (transformants per recipient) under optimised conditions, which is expected to drop even further to a minimum of 10^-16 due to soil conditions and a lowered concentration of DNA available to cells. Previous studies have shown that chromosomal DNA released to soil is only available to A. calcoaceticus for limited period of time and that A. calcoaceticus does not maintain detectable competence in soil. Taken together, these results suggest that A. calcoaceticus does not take up non-homologous plant DNA at appreciable frequencies under natural conditions. Received: 1 November 1996 / Accepted: 18 April 1997     

NewAgrobacterium helper plasmids for gene transfer to plants

We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these disamed Ti plasmids for plant transformation viaAgrobacterium are discussed.     

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T₁ progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.     

植物转基因方法概述

综述了植物基因转化的不同方法。植物基因转化方法主要包括直接基因转移和农杆菌介导的转化,直接基因转移又包括PEG介导、电激介导和基因枪方法。相比之下,农杆菌转化方法具有明显的优势;本文对其方法和机制作了较为全面的介绍。     

Predictive modeling of non‐viral gene transfer

In non‐viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine‐ and PEI‐mediated exogenous gene expression distributions from single cell time‐lapse analysis. Broad Poisson‐like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co‐transfection analysis with YFP‐ and CFP‐coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two‐step process is assumed, with the first being the low‐probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non‐viral vectors. Biotechnol. Bioeng. 2010. 105: 805–813. © 2009 Wiley Periodicals, Inc.     

Horizontal gene transfer of a genetic island encoding a type III secretion system distributed in Vibrio cholerae

Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS‐related genes were distributed among the various serogroups and pulsed‐field gel electrophoresis of NotI‐digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS‐related genes had similar patterns. Additionally, naturally competent T3SS‐negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS‐related genes occurs among V. cholerae in natural ecosystems.