Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells.

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Introduction of liposome-encapsulated SV40 DNA into cells

DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.     

Inhibitors of ADP-ribosyl transferase enhance the transformation of NIH3T3 cells following transfection with SV40 DNA

The influence of inhibitors of the enzyme ADP-Ribosyl Transferase (ADPRT) upon the morphological transformation of NIH3T3 cells by calcium phosphate-SV40 DNA co-precipitates was examined. Marked enhancement in the frequency of foci formation was noted when 3-methoxybenzamide was added to cells either during or after exposure to SV40 DNA, but there was no effect when cells were only pretreated with the inhibitor. The greatest enhancement was observed when cells were exposed to inhibitor both during and after transfection with DNA. The involvement of ADPRT activity in the events during transfection of DNA into cells is not understood. These results, however, highlight the importance of the key regulatory molecule poly (ADP-ribose) in the events which occur within the nucleus and which lead to transformation with SV40 DNA.     

High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.     

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.     

Apurinic sites cause mutations in simian virus 40

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.     

Transfection of embryonal carcinoma cells at high efficiency using liposome-mediated transfection

Embryonal carcinoma (EC) cells are recognized as an excellent model system for studying the early stages of mammalian development. Many studies performed with EC cells involve transient transfection with promoter/reporter gene constructs and/or mammalian expression vectors. One of the limitations of working with EC cells is their inability to be transfected at high efficiency. In most cases, EC cells are transfected using the calcium phosphate method. The objective of this study was to identify protocols and culture conditions that significantly increase the transfection efficiency of EC cells. F9 EC cells were used for this purpose, because they are the EC cell line studied most commonly. We show that the transfection efficiency of F9 EC cells using the calcium phosphate method is less than 5%; whereas, their transfection efficiency can be improved approximately 15-fold using optimized culture conditions and liposome-based transfection reagents. Specifically, we demonstrate that more than 50% of F9 EC cells can be transfected using LipofectAMINE 2000. In addition to higher levels of transfection, there is much less plate-to-plate variation with liposome-based reagents as compared to transfection with calcium phosphate. Interestingly, transfection efficiency using these reagents was found to be inversely related to cell density. This contrasts sharply with the recommendation that transfection with LipofectAMINE 2000 or LipofectAMINE in conjunction with the PLUS reagent be performed at high cell densities. Given the improvements in transfection efficiency reported here, it will now be possible to perform studies with F9 EC cells that require transfection at significantly higher levels than that achieved using the calcium phosphate method. Overall, the highest transfection efficiencies were consistently obtained using LipofectAMINE 2000.     

Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.     

Transfection Alters Ion Transport in MDCK Cells

In the course of an investigation into the effect of Tamm-Horsfall protein (THP) on ion transport, we performed stable transfection of THP into MDCK cells using the SV40 or the cytomegalovirus (CMV) promoter. As controls, we transfected MDCK cells with an ``empty' plasmid containing SV40 or CMV promoter but without THP cDNA. In another set of controls, we subjected cells to transfection procedures without DNA (mock transfection). K influx was not altered in cells subjected to mock transfection procedures without DNA, but both ouabain sensitive (OS) and ouabain resistant (OR) components of K influx were diminished in cells transfected with THP cDNA using either SV40 or CMV promoter. However, K influx was also reduced in cells transfected with a control plasmid containing either the SV40 promoter alone, or the CMV promoter alone, without the THP cDNA. Thus, the transport alterations were caused by transfection and not by THP. The reduction in ouabain-sensitive K influx was accompanied by a proportional reduction in the abundance of Na-K pump units as assessed by [³H] ouabain binding. [³H] bumetanide binding, a measure of the number of functioning NaK2Cl cotransporter sites, was reduced pari passu with the reduction in bumetanide-sensitive K influx. These results highlight the possibility that alterations in properties of transfected cells may not be solely due to the presence of transfected protein, but the result of some process associated with transfection itself. Without appropriate controls to evaluate this possibility, results of transfection studies are subject to potentially faulty and misleading interpretation. Received: 25 April 1995/Revised: 25 September 1995     

Tracking the pathway of calcium phosphate/DNA nanoparticles during cell transfection by incorporation of red-fluorescing tetramethylrhodamine isothiocyanate–bovine serum albumin into these nanoparticles

Calcium phosphate nanoparticles were prepared by precipitation from water and were then functionalized by DNA. These particles are taken up by living cells and function as gene transfer agents, i.e., the DNA is brought into a cell’s nucleus and is incorporated there into the cell’s genome (transfection). DNA which encodes for enhanced green fluorescent protein leads to green fluorescence of successfully transfected cells. By adding the red-fluorescing marker tetramethylrhodamine isothiocyanate–bovine serum albumin (TRITC-BSA) to the nanoparticles, their pathway into the cell and within the cell could be followed by fluorescence microscopy. A clear correlation between the uptake of nanoparticles and the efficiency of transfection was found. Aggregates of DNA/TRITC-BSA alone were not able to enter the cells, i.e., the inorganic nanoparticles are necessary as a carrier through the cell membrane.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

钠米颗粒介导质粒DNA转染体外真核细胞

DNA传递是基因表达与功能研究及其医学应用的重要技术,安全高效的DNA传递一直是研究者期待的目标。利用一种新的阳离子多聚物脱乙酰甲壳胺16介导重组质粒pcDNA3vp1转染COS7细胞,RTPCR可检测到目的基因vp1在mRNA水平的表达,实时定量PCR结果表明其转染效率介于脂质体与磷酸钙法之间,同时还对转染条件进行了探讨。DNA结合分析发现脱乙酰甲壳胺16能够与DNA形成核酸纳米颗粒,提高DNA稳定性,促进真核细胞转染效率的提高。这些结果表明脱乙酰甲壳胺16确能做为一种新型的非病毒纳米DNA传递载体,并将可能在基因表达与功能研究及基因治疗等领域发挥重要作用 。     

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.     

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.     

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGNE 6 transfection technology. The nocovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a mximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G₂/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.     

SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T-antigen

A modification of the Graham and Van der Eb (1974) DNA-calcium phosphate coprecipitation technique is shown to routinely transfect 15% of CV-1 cells with SV40 DNA. The transfection is done in suspension after detachment of cells by trypsin digestion. Transfection efficiency was measured by staining cells for the presence of SV40 T-antigen by indirect immunofluorescence and by assaying for the presence of SV40 early message by the Berk and Sharp (1978) technique.     

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.     

Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants

In vitro matured bovine oocytes at the metaphase-II stage were slowly frozen in phosphate buffered saline (PBS) containing 1.0 M glycerol, 1.0 M dimethylsulfoxide (DMSO) or 1.0 M propylene glycol (PROH). When thawed rapidly, more (P<0.05) oocytes were morphologically normal after being frozen with DMSO (86%) or PROH (83%) than with glycerol (62%). When inseminated in vitro with frozen-thawed bull spermatozoa, higher (P<0.05) penetration rates were observed in DMSO (79%) or PROH (76%) than in glycerol (48%). The percentages of oocytes developing to the 2-cell stage at 48 h postinsemination were also significantly (P<0.05) higher in DMSO (51%) and PROH (54%) than in glycerol (33%). However, a significant increase in the proportions of 8-cell embryos (46 vs 21 to 26%; P<0.05) at 72 h postinsemination and morulae (14 vs. 6 to 8%; P<0.05) was derived from oocytes frozen with PROH than with DMSO or glycerol. In conclusion, the type of cryoprotectant used is one of the critical factors affecting developmental competence of bovine oocytes frozen at the metaphase-II stage. For this stage of oocytes, PROH was the most effective, yielding a large number of 8-cell embryos and morulae than either glycerol or DMSO in a slow freezing method combined with a 3-step thawing protocol.     

Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX

Objectives

To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.

Results

Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.

Conclusion

Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.