Comparison of plasmid DNA versus PCR amplified gene of insert DNA for nucleofection in Kasumi-1 cells

Plasmid electroporation, or its optimized version nucleofection, is an important technique for gene transfection of cells in suspension. However, substantial cell death and/or low transfection efficiency are still common for some cell lines. By using enhanced green fluorescent protein (EGFP) as a reporter, we compared the use of PCR amplified EGFP (PaEGFP) and its parental plasmid (pEGFP-N2) for nucleofection in Kasumi-1 cells. We found that PaEGFP induced significantly lower cell death but had similar transfection efficiency compared to its parent plasmid (pEGFP-N2). Most importantly, contrary to the pEGFP-N2-nucleofected cells, the PaEGFP-nucleofected cells subsequently grew properly. Tests in other cell lines also implied that PaEGFP indeed induced consistently less cell death, but transfection efficiencies varied, being good in suspension cell lines but lower in adhesive cell lines. We suggest that direct transfection with PCR amplified genes can be a simple and useful approach for optimization of electropulse-based transfection not only of Kasumi-1 cells, but also may be useful for other cell lines that are difficult to transfect in suspension.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9683-y) contains supplementary material, which is available to authorized users.     

Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines

Loss of a functional mismatch repair (MMR) system in colorectal cancer (CRC) cells is associated with microsatellite instability and increased sensitivity to topoisomerase inhibitors. In this study, we have investigated whether a defect in double-strand break (DSB) repair by non-homologous end-joining (NHEJ) could explain why MMR-deficient CRC cells are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor. To evaluate the efficiency and the fidelity of DSB repair, we have transiently transfected plasmids containing cohesive or non-complementary ends in cells with various MMR defects. We have observed that the repair efficiency of DSB with cohesive and non-complementary ends is comparable in all cell lines. In contrast to the MMR-proficient cell line HT29, the MMR-deficient cell lines were highly accurate in repairing DSB with cohesive ends, but this characteristic could not be directly assigned to the primary MMR deficiency. Furthermore, CPT treatment had no detectable effect on the repair of cohesive ends but significantly decreased the repair efficiency of non-complementary DSB. In conclusion, although our observations show that DSB repair efficiency by NHEJ decreases upon treatment with CPT, which possibly contributes to its cytotoxicity, it is quite unlikely that it accounts for the hypersensitivity of MMR-deficient cells to topoisomerase inhibitors.     

Interplay between Cernunnos-XLF and nonhomologous end-joining proteins at DNA ends in the cell

Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends.     

DNA double-strand break repair in eukaryotic cell lines having radically different radiosensitivities

TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double-strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. We have therefore compared the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with 137Cs gamma rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy is similar for both cell lines with 50% of the rejoining completed in about 12 min. Approximately 83 and 87% of the DSB are rejoined in the TN-368 and V79 cells, respectively, by 1 h postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 h postirradiation, whereas approximately 92% of the DSB are rejoined in the V79 cells by 2 h postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for both cell lines, but this relationship is not as pronounced for the TN-368 cells. In general, these findings do not support the hypothesis that unrejoined DNA DSB represent the critical molecular lesion responsible for cell death.     

Correct evaluation of reporter assays in different cell lines by direct determination of the introduced plasmid amount

Transfection efficiency in reporter gene assays is usually determined by cotransfection of a reference reporter gene under the control of a constitutively active strong promoter and determination of the reference enzyme activity. The SV40 promoter-driven beta-galactosidase reporter plasmid is frequently used as the reference reporter plasmid. Here we show that the beta-galactosidase expression in different cell lines does not correctly reflect the amount of plasmid taken up by cells and thus is not an accurate measure of transfection efficiency. The direct determination of introduced plasmid concentration in lysates of transfected cells is suitable for monitoring the transfection efficiency in reporter gene assays even if different cell lines are compared.     

Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA

Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector.     

Human DNA ligases I and III have stand-alone end-joining capability,but differ in ligation efficiency and specificity

Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3′-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5′-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg²⁺, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg²⁺ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.     

Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli

Cell death may occur by either of two mechanisms: apoptosis or necrosis. Necrosis, the first type of cell death to be recognized, is an uncontrolled degenerative phenomenon invariably caused by noxious stimuli and is the result of irreversible failure of membrane function. Apoptosis, on the other hand, is a death process which involves a series of well-organized events which require active cell participation, and is primarily caused by physiological stimuli. In the present study we show that cell death induced by a range of varied agents may take the form of either apoptosis or necrosis. Apoptotic cell death was found to occur at low levels of these agents, while at higher levels necrosis occurred. Hence, cells which are not killed directly, but merely injured by these agents, have the capacity to activate an internally programmed suicide death mechanism, whereas cells receiving greater injuries apparently do not. In addition, the presence of extracellular calcium was found to be necessary for the induction of apoptosis with all agents tested.     

A minimal cytomegalovirus intron A variant can improve transgene expression in different mammalian cell lines

The expression enhancement by cytomegalovirus promoter and different intron A (IA) variants were evaluated in CHO-K1, HepG2, HEK-293 and COS-7 cells by assessing the levels of luciferase activity. This data along with mRNA levels measurement indicated that the construct harboring an IA variant with a 200-nucleotide deletion (Δ200) had the greatest impact on increasing luciferase expression among all constructs evaluated. Based on these results, we redesigned pCMV–IA variants and cloned them into plasmids expressing a humanized antibody. These plasmids were then used to transfect CHO-K1 cells. Production of the antibody was not augmented with the Δ200 promoter variant. The 600-nucleotide deletion (Δ600) and whole IA promoter variants expressed similar levels of the recombinant protein. These data indicate that the IA-based enhanced expression of transgenes depends on a small region within the intron.     

Single-strand breaks can lead to complex configurations of plasmid DNA in vitro

DNA species which migrate extremely slowly in agarose gel electrophoresis may be formed from plasmid DNA containing radiation-induced single-strand breaks (ssbs). Postirradiation heat treatment in low ionic strength buffer and subsequent incubation with Mg2+ strongly enhanced the formation of these species. Electron micrographs taken after such treatment show numerous complex configurations containing DNA material from several plasmid molecules. Less extreme formation of slowly migrating DNA occurred without postirradiation heat treatment or Mg2+ incubation when the DNA was co-precipitated with calcium phosphate in a physiologically balanced buffer and incubated under conditions used for DNA-mediated gene transfer. The data suggest that homologous pairing between single-stranded regions formed in relation to ssbs may contribute to cohesion between different molecules. The significance of the cohesion process for gene transfer experiments and cellular radiation effects is discussed.     

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.     

Restriction endonuclease sensitivity of DNA containing globin genes in different murine erythroleukemia cell lines

The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSo-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with alpha-globin cDNA but not with the beta-globin cDNA, suggesting either rearrangement or deletion of an alpha-globin gene-like fragment in DR10 DNA.     

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.     

Generation of tobacco lines with widely different reduction in nicotine levels via RNA silencing approaches

Issues related to the nicotine content of tobacco have been public concerns.Several reports have described decreasing nicotine levels by silencing the putrescine N-methyltransferase (PMT) genes, but the reported variations of nicotine levels among transgenic lines are relatively low in general. Here we describe the generation in tobacco (Nicotiana tabacum) lines with widely different, reduced nicotine levels using three kinds of RNA-silencing approaches.The relative efficacies of suppression were compared among the three approaches regarding the aspect of nicotine level in tobacco leaves.By suppressing expression of the PMT genes, over 200 transgenic lines were obtained with nicotine levels reduced by 9.1-96.7%. RNA interference (RNAi) was the most efficient method of reducing the levels of nicotine,whereas cosuppression and antisense methods were less effective. This report gives clues to the efficient generation of plants with a variety of metabolite levels, and the results demonstrate the relative efficiencies of various RNA-silencing methods.     

Energy-structure correlations of plasmid DNA in different topological forms.

Differential scanning microcalorimetry (DSC), UV absorption and circular dichroism (CD) have been used to study structure and stability of linear (lin), open circular (oc), supercoiled (cd) and relaxed circular duplex (rd) DNA and calf thymus (CT) DNA. Investigations were made in low salt buffer and in the presence of 7.2 M NaClO4. The chaotropic action of perchlorate promotes a reduction of the overall stability of DNA, which permits a direct determination of the transition enthalpies of all four DNA configurations. The stabilities against thermal denaturation have been found to increase in the series lin approximately oc less than cd less than rd. These relative stabilities can be rationalized on the basis of the linkage between supercoiling and secondary structural changes in topologically constrained duplex DNA. On the basis of these studies, a model of the melting process could be suggested that is consistent with the energetic and spectroscopic data.     

DNA methylation and chromosome instability in lymphoblastoid cell lines

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.