Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.

The technique of DNA transfer by electroporation was investigated in an effort to evaluate its utility for the identification of developmentally controlled regulatory sequences. Transient and stable gene expression was detected in a variety of lymphoid cell lines subjected to electroporation. No correlation existed between the levels of chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) expression and stable transfection frequency. In all lymphoid cell lines tested, the simian virus 40 early region was a better promoter than was the Rous sarcoma virus long terminal repeat.     

Optimisation and evaluation of a high-throughput mammalian protein expression system

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.     

GFP-Human high-affinity carnitine transporter OCTN2 protein: subcellular localization and functional restoration of carnitine uptake in mutant cell lines with the carnitine transporter defect.

Individuals with the plasmalemmal high-affinity carnitine transporter defect present with progressive infantile-onset carnitine-responsive cardiomyopathy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopathy, and failure to thrive. The carnitine uptake defect (CUD) has been documented in their cultured skin fibroblasts, lymphoblasts, and/or myoblasts. The cDNA encoding the high-affinity sodium-dependent human carnitine transporter OCTN2 has recently been cloned. We used the green fluorescent protein (GFP) as a living marker for positively transfected cells in our expression studies of the high-affinity carnitine transporter OCTN2 cDNA in cell lines with the CUD. Transfection of cell lines from 12 unrelated patients (nine fibroblast and three lymphoblastoid) with a GFP construct harboring the wild-type full-length OCTN2 cDNA was done using LipoTAXI. Transient and stable expression of the recombinant GFP-human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the fibroblast and stable transfection of the lymphoblastoid cell lines were achieved. There was functional restoration of carnitine uptake in the transfected mutant cell lines, thereby confirming the identity of the transfected cDNA. In addition, we report the first demonstration of the subcellular localization of an in-frame fusion GFP-human high-affinity carnitine transporter OCTN2 protein in the plasma membrane by confocal laser-scanning fluorescence microscopy.     

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.     

Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F₁ generation.     

Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaCl tolerant cell lines

Selection of stable, NaCl tolerant alfalfa (Medicago sativa L.) cell lines was accomplished by a step-up selection procedure, whereby cell lines originally selected for tolerance at 0.5% NaCl were subsequently selected at 1.0% NaCl. Sodium chloride tolerant cell lines retained tolerance following four subcultures (16 weeks) on control media (0% NaCl). Plants were regenerated from selected NaCl tolerant cell lines of three initial genotypes, one diploid (2n=2x=16) and two tetraploids (2n=4x=32). In addition, plants were regenerated from control cell lines maintained on 0% NaCl media for the same duration. Plants regenerated from NaCl tolerant cell lines were characterized by extensive somaclonal variation compared to plants regenerated from control lines. Morphologically, all plants regenerated from NaCl tolerant cell lines are abnormal and many (44.7%) were extreme dwarfs (maximum height of 5 cm). The grossly aberrant phenotypes prevented an in-depth characterization of many of the plants regenerated from NaCl tolerant cell lines. Most plants regenerated from NaCl tolerant cell lines had unbalanced polyploid chromosome sets with the most extreme cytogenetic variant having 106 chromosomes. In contrast, 98.5% of the plants regenerated from control cell lines were euploid (85% were tetraploid, 15% were octoploid). Isozyme phenotypes of the plants from NaCl tolerant cell lines were also extensively altered, compared to plants from control cell lines. In vitro NaCl tolerance was maintained following plant regeneration for nine of the 12 regenerants tested. Importantly, whole plant NaCl tolerance was expressed in two of the seven regenerated plants tested at the whole plant level; however, only one of these plants has flowered and is both male and female sterile; the other plant has never flowered. Although NaCl tolerant alfalfa cell lines are efficiently selected, the extensive somaclonal variation that accompanied the selection was a deterrent to successful recovery of heritable NaCl tolerance.     

High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures

Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment, was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999     

Hygromycin B-selected cell lines from GAL4-regulated pUAST constructs

Here we report the generation of stable, selectable Drosophila S2 cell lines using the UAS-GAL4 system. Cloning of the hygromycin resistance gene into the pUAST vector and cotransfection with other pUAST constructs in S2 cells results in coexpression of up to four different proteins under hygromycin selection. Protein expression is driven by the ubiquitous Actin5C-GAL4 driver and cell cultures are maintained in hygromycin-supplemented, serum-free media to ensure constitutive protein production. Visual comparison of cells cotransfected with GFP and RFP demonstrates a uniform cell population expressing both markers simultaneously, while Western blot analysis shows concurrent expression of MYC3-tagged proteins. In addition, fluorescent cell sorting (FACS) analysis shows that 80% of the total cell population express the GFP marker. Our data indicate that using this technique it is possible to establish stable, selectable cell lines that provide a pool of readily accessible protein. This facilitates protein-based studies and abolishes the need to carry out time-consuming and expensive transfections.     

Antibody production by molecular farming in plants

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality.     

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment

The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene ( aphIV) and the unselected reporter gene ( gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T(0)), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T(0) plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T(0)) and their progeny (T(1)): (1) absence of transgene expression in T(0) plants (41% of lines), (2) sterility of T(0) plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.     

Comparison of transient protein expression in tobacco leaves and plant suspension culture

Transient gene expression is being developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. Transient expression of the gus-intron reporter gene was facilitated in three different tobacco species. Two different approaches to T-DNA delivery were compared: (1) infiltration of a prototrophic strain of Agrobacterium into leaves and (2) coculture of plant cell suspension cultures with an Agrobacterium auxotroph. Wounding of plant tissues with a wire brush prior to infiltration had a large positive impact on Nicotianabenthamiana leaves but not for Nicotiana tabacum or Nicotiana glutinosa. The best expression level achieved by leaf infiltration was in N. benthamiana (0.025% total soluble protein). A cell suspension culture line of N. glutinosa achieved an expression level greater than 0.04% TSP. The tissue culture-based technique therefore provides improved levels of transient expression under aseptic conditions to facilitate improvements in expression by control of the plant cell culture and Agrobacterium coculture environments.     

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.     

Subpopulations of fibroblasts from mouse skeletal muscle defined by clonal variation for 5' nucleotidase expression

A primary cloning technique has been employed for the isolation of nine spontaneously transformed cell lines from mouse skeletal muscle. Four of these lines were isolated after selection for partial resistance to the purine (adenine) analog 2'6'diaminopurine and five were isolated from non-selected control dishes. Four of the nonselected lines and three of the selected lines demonstrated a fibroblastoid morphology in vitro. The other two cell lines (one from each group) were epithelioid. Two of the three selected fibroblastoid lines were found to contain significant quantities of the enzyme 5'nucleotidase (EC3.1.3.5), whereas the four nonselected fibroblastlike lines, one selected fibroblastlike line, and the two epithelioid lines did not. In the two cell lines expressing 5'nucleotidase activity, this expression was stable in the absence of selective pressure. Histochemical staining of mouse skeletal muscle for 5'nucleotidase activity demonstrated positive staining in the cells of small blood vessels and in a subset of the connective tissue cells. The bulk of the skeletal muscle tissue, however, had no detectable 5'nucleotidase activity. We propose that the two cultivatable types of fibroblastoid cell lines represent distinct classes of fibroblastlike cells in vivo, reflecting alternative states of stable cellular differentiation involving 5'nucleotidase expression.     

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.     

miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.     

Visualization of PtdIns3P dynamics in living plant cells

To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.     

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY‐2) suspension cells

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease‐deficient tobacco BY‐2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY‐2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full‐length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV‐1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four‐fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N‐terminal sequencing data revealed that the antibody has two cleavage sites within the CDR‐H3 and one site at the end of the H4‐framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.