Targeted correction of single-base-pair mutations with adeno-associated virus vectors under nonselective conditions

Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.     

Involvement of Cellular Double-Stranded DNA Break Binding Proteins in Processing of the Recombinant Adeno-Associated Virus Genome

Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.     

Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.

Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy.     

Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells

Intestinal gene transfer offers promise as a therapeutic option for treatment of both intestinal and non-intestinal diseases. Recombinant adeno-associated virus serotype 2, rAAV2, based vectors have been utilized to transduce lung epithelial cells in culture and in human subjects. rAAV2 transduction of intestinal epithelial cells, however, is limited both in culture and in vivo. Proteasome-inhibiting agents have recently been shown to enhance rAAV2-mediated transgene expression in airway epithelial cells. We hypothesized that similar inhibition of proteasome-related cellular processes can function to induce rAAV2 transduction of intestinal epithelial cells. Our results demonstrate that combined treatment with proteasome-modulating agents MG101 (N-acetyl-L-leucyl-L-leucyl-L-norleucine) and Doxorubicin synergistically induces rAAV2-mediated luciferase transgene expression by >400-fold in undifferentiated Caco-2 cells. In differentiated Caco-2 monolayers, treatment with MG101 and Doxorubicin induces transduction preferentially from the basolateral cell surface. In addition to Caco-2 cells, treatment with MG101 and Doxorubicin also results in enhanced rAAV2 transduction of HT-29, T84, and HCT-116 human intestinal epithelial cell lines. We conclude that MG101 and Doxorubicin mediate generic effects on intestinal epithelial cells that result in enhanced rAAV2 transduction. Use of proteasome-modulating agents to enhance viral transduction may facilitate the development of more efficient intestinal gene transfer protocols.     

Major subsets of human dendritic cells are efficiently transduced by self-complementary adeno-associated virus vectors 1 and 2

Dendritic cells (DC) are antigen-presenting cells pivotal for inducing immunity or tolerance. Gene transfer into DC is an important strategy for developing immunotherapeutic approaches against infectious pathogens and cancers. One of the vectors previously described for the transduction of human monocytes or DC is the recombinant adeno-associated virus (rAAV), with a genome conventionally packaged as a single-stranded (ss) molecule. Nevertheless, its use is limited by the poor and variable transduction efficiency of DC. In this study, AAV type 1 (AAV1) and AAV2 vectors, which expressed the enhanced green fluorescent protein and were packaged as ss or self-complementary (sc) duplex strands, were used to transduce different DC subsets generated ex vivo and the immunophenotypes, states of differentiation, and functions of the subsets were carefully examined. We show here for the first time that a single exposure of monocytes (M(o)) or CD34(+) progenitors (CD34) to sc rAAV1 or sc rAAV2 leads to high transduction levels (5 to 59%) of differentiated M(o)-DC, M(o)-Langerhans cells (LC), CD34-LC, or CD34-plasmacytoid DC (pDC), with no impact on their phenotypes and functional maturation of these cells, compared to those of exposure to ss rAAV. Moreover, we show that all these DC subpopulations can also be efficiently transduced after commitment to their differentiation pathways. Furthermore, these DC subsets transduced with sc rAAV1 expressing a tumor antigen were potent activators of a CD8(+)-T-cell clone. Altogether, these results show the high potential of sc AAV1 and sc AAV2 vectors to transduce ex vivo conventional DC, LC, or pDC or to directly target them in vivo for the design of new DC-based immunotherapies.     

Comparison of non-viral transfection methods in melanoma cell primary cultures

Melanoma primary cultures were transiently transfected via electroporation and lipofection for comparison. Transfection efficiency was superior with electroporation (58+/-9%) as compared to lipofection (23+/-9%) as determined by enhanced green fluorescent plasmid (EGFP) transfection. Secretion of IL-2 persisted for up to 3 weeks after electroporation. The increase in sensitivity against immunologic effector cells by transfection with IL-2 was not significant. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction.     

Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with beta-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 x 10(12) vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression.     

Kinetics of Efficient Recombinant Adeno-Associated Virus Transduction in Retinal Pigment Epithelial Cells

The aim of this study was to investigate the premise that retinal pigment epithelial (RPE) cells are more permissive to recombinant adeno-associated virus (rAAV) transduction than other cells. We investigated the kinetics and mechanisms of rAAV transduction in RPE cells and found that the transduction efficiencies of cultured RPE cells HRPE51 and ARPE19 were significantly higher than those of 293 (P < 0.008) and HeLa (P < 0.025) cells. In addition, RPE cells reached maximum transduction efficiency at a much lower m.o.i. (m.o.i. 10) than 293 cells (m.o.i. 25). Competition experiments using 1 microg/ml heparin inhibited the high level of transduction in RPE cells by 30%, but additional heparin failed to reduce rAAV transduction further. Southern hybridization of low-molecular-weight DNA from transduced RPE cells indicated that 42% of single-stranded rAAV DNA was translocated into the nucleus by 2 h postinfection. By 6 h postinfection, double-stranded rAAV DNA was observed, which coincided with the onset of transgene expression. Southern and fluorescence in situ hybridization of total genomic DNA indicated that long-term transgene expression in RPE cells was maintained by the integration of rAAV into the cellular chromosome. Together, these results suggest that the high permissiveness of RPE cells is not related to the presence of heparan sulfate receptors or nuclear trafficking but may be due to an enhanced rate of second-strand synthesis and that integration in RPE cells is responsible for long-term transgene expression.     

Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.     

Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia

Recombinant adeno-associated virus type 5 (rAAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.     

Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.     

Impact of transduction towards the proliferation and migration as well as the transduction efficiency of human umbilical cord-derived late endothelial progenitor cells with nine recombinant adeno-associated virus serotypes

Objectives

To evaluate the transduction efficiency of human umbilical cord-derived, late endothelial progenitor cells late (HUCB-late EPCs) with nine recombinant adeno-associated virus (rAAV) serotypes and the ability of proliferation and migration of the cells after transduction.

Results

rAAV2 and rAAV6 showed a greater ability than other serotypes to transduce late EPCs (P < 0.05). After transduction, cell proliferation ability weakened (P < 0.05), but the ability of migration to stromal cell-derived factor (SDF-1) unchanged.

Conclusion

There is an advantage of choosing the optimal rAAV serotype as a gene vector to alter the biologic characteristics of late EPCs.

     

Kinetics of recombinant adeno-associated virus-mediated gene transfer

Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to beta-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 microgram of hFIX/10(6) cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.     

Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency

Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 microg DNA for 10 x 10(6) cells via electroporation which resulted in 10-20 positive cells/mm2, M9-assisted lipofection of 2 x 10(5) cells with 2 microg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 x 10(6) ES cells and 6 microg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.     

重组腺相关病毒转染神经干细胞球的实验研究

目的:探讨重组腺相关病毒2型(rAAV2)对神经干细胞球的转染能力.方法:①将FITC标记的rAAV2(FITC-rAAV2)分成两组,A组直接与神经干细胞球混合,B组与肝素混匀后再与神经干细胞球混合,孵育30 min后在荧光显微镜下观察;②含有GFP报告基因的rAAV2(rAAV2-GFP)与神经干细胞球孵育30 min后,分成两组:A组继续在培养箱内培养,B组分散成单细胞后移植到大鼠脑内,一个月后分别在荧光显微镜下观察神经干细胞球和大鼠脑组织切片中报告基因的表达情况;③将含有低氧启动子(低氧应答元件,HRE)、VEGF和GFP的rAAV2(rAAV2-HRE-VEGF-GFP)转染神经干细胞球后分为两组:A组在低氧条件下培养,B组在常规条件下培养,72 h后观察报告基因的表达情况.结果:①FITC-rAAV2转染神经干细胞球的结果:A组有明亮的绿色荧光,B组基本无绿色荧光;②rAAV2-GFP转染神经干细胞球后一个月,A、B两组均可以看到绿色荧光;③rAAV2-HRE-VEGF-GFP转染神经干细胞球后72 h,A组可见绿色荧光,B组无绿色荧光.结论:rAAV2可以与神经干细胞球特异性结合,rAAV2携带的外源基因在体内和体外均可以有效表达,rAAV2携带外源基因的表达可以人为调控.     

Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes

The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7–78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6–12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes.     

In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8

BACKGROUND: Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B(12), adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). METHODS: The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 x 10(10) and 1 x 10(11) particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. RESULTS: Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken beta-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. CONCLUSIONS: These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.     

Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.