DNA Transfection Method Research Articles

A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9 System.

The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.

Looking Back, Moving Forward: Lipid Nanoparticles as a Promising Frontier in Gene Delivery.

Lipid nanoparticles (LNPs) have shown remarkable success in delivering genetic materials like COVID-19 LNP vaccines, such as mRNA-1273/SpikeVax by Moderna and BNT162b2/Comirnaty by BioNTech/Pfizer, as well as siRNA for rare inherited diseases, such as Onpattro from Alnylam Pharmaceuticals. These LNPs are advantageous since they minimize side effects, target specific cells, and regulate payload delivery. There has been a surge of interest in these particles due to their success stories; however, we still do not know much about how they work. This perspective will recapitulate the evolution of lipid-based gene delivery, starting with Felgner's pioneering 1987 PNAS paper, which introduced the initial DNA-transfection method utilizing a synthetic cationic lipid. Our journey takes us to the early 2020s, a time when advancements in bionano interactions enabled us to create biomimetic lipoplexes characterized by a remarkable ability to evade capture by immune cells in vivo. Through this overview, we propose leveraging previous achievements to assist us in formulating improved research goals when optimizing LNPs for medical conditions such as infectious diseases, cancer, and heritable disorders.

Enhancement of drug electrotransfer by extracellular plasmid DNA

Electroporation is a widely established method for molecular delivery across electric field perturbed plasma membrane. It can be used as a non-viral DNA transfection method, or as a way to achieve small molecule delivery to or extraction from cells. We examined the possibility of combining the DNA delivery to the cells with small molecule transport across electroporated plasma membrane. The results show that the presence of DNA in electroporation medium increases the extraction of fluorescent dye calcein from calcein-AM loaded cells as well as the delivery of small-molecule drug bleomycin to the cells. We propose that these results may have implications in enhanced drug delivery using electroporation both in vivo and in clinics.

A method for high transfection efficiency in THP-1 suspension cells without PMA treatment

Adherent cells such as mouse RAW cells or human cancer U2OS cells are beneficial to DNA transfection, with 20%–60% transfection efficiency. However, this DNA transfection is rarely used on suspension cells due to its low transfection efficiency (≤5%). We recently found a new DNA transfection method to increase the efficiency up to 13.5% in suspension cells without PMA treatment. We also found that DNA transfection of human TNFAIP1 or CXCL1 recombinant plasmid DNA in THP-1 cells induces a high level of TNF-α protein. Overall, this new method is simple yet efficient and can be used for the overexpression of DNA in suspension cells.

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction.Keywords: AAV, serotype DJ/8, GFP, Hepatocytes

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents.

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-016-9673-5) contains supplementary material, which is available to authorized users.

Anti-desmocollin autoantibodies in nonclassical pemphigus.

Despite the established pathogenic role of anti-desmoglein (Dsg) antibodies in classical pemphigus, the significance of autoantibodies to another desmosomal cadherin, desmocollin (Dsc) is at present unknown. No consistent immunoassay for immunoglobulin (Ig) G autoantibodies to Dscs has been developed. The aim of this study was to develop reliable assays to detect anti-Dsc autoantibodies. We expressed soluble recombinant proteins (RPs) of human Dsc1-3 in mammalian cells and examined sera of various types of pemphigus, including 79 paraneoplastic pemphigus (PNP) sera, by novel enzyme-linked immunosorbent assays (ELISAs) using the RPs. We also performed ELISAs of Dsc baculoproteins and used the complementary DNA (cDNA) transfection method, and compared the results with those of mammalian ELISAs. Through mammalian ELISAs, IgG autoantibodies to Dsc1, Dsc2 and Dsc3 were detected in 16.5%, 36.7% and 59.5% of PNP sera, respectively, and considerable numbers of pemphigus herpetiformis (PH) and pemphigus vegetans (PVeg) sera reacted strongly with Dsc1 and Dsc3. Mammalian ELISAs were highly specific and more sensitive than baculoprotein ELISAs or the cDNA transfection method. Several Dsc-positive sera, particularly PH sera, showed no reactivity with Dsgs. The reactivity of PNP serum and PVeg serum with Dscs was not abolished by pre-absorption with Dsg RPs. The results of these novel ELISAs indicated that IgG anti-Dsc autoantibodies were frequently detected and potentially pathogenic in nonclassical pemphigus.

Sandwiching spherical 1,2-dioleoyltrimethylammoniumpropane liposome in gold nanoparticle on solid transducer for electrochemical ultrasensitive DNA detection and transfection

Cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium propane (DOTAP) liposome is spherically sandwiched in gold nanoparticle (abbreviated as sDOTAP–AuNP) onto a gold electrode surface. The sDOTAP–AuNP is applied for electrochemical label free DNA sensing and Escherichia coli cell transfection for the first time. Complementary target (named as hybridized), non-complementary target (un-hybridized) and single base mismatch target (named as SMM) hybridized surfaces are discriminated sensitively and selectively in presence of [Fe(CN)6]3−/4−. Double strand specific intercalator methylene blue in combination with [Fe(CN)6]3− is used to enhance target detection limit down to femtomolar concentration. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) techniques are used for characterizing DNA sensing. High Resolution Transmission Electron Microscopy (HRTEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) techniques are used to confirm the spherical nature of the sDOTAP–AuNP–DNA composite in solution and on the solid surface. DNA on the sDOTAP–ssDNA is transferred by potential stripping method (+0.2V (Ag/AgCl)) into buffer solution containing E. coli cells. The transfection is confirmed by the contrast images for the transfected and non-transfected cell from Confocal Laser Scanning Microscopy (CLSM). The results demonstrate effectiveness of the electrochemical DNA transfection method developed and could be applied for other cells.

Two Cases of Pemphigus Vegetans With IgG Anti–Desmocollin 3 Antibodies

Pemphigus vegetans shows clinically vegetating and/or pustular skin lesions mainly on the intertriginous areas and histopathologically neutrophilic and eosinophilic pustules in the epidermis. Pemphigus vegetans shows IgG reactivity mainly with desmoglein (Dsg) 3, but also with other autoantigens, including Dsg1 and desmocollins (Dscs). We examined antigen profiles in 2 cases of pemphigus vegetans. (1) A women in her 80s presented with typical vegetating skin lesions on the right inguinal region with typical histopathological features. Immunoblotting using normal human epidermal extracts detected IgG antibodies to Dsg1 and Dscs. Enzyme-linked immunosorbent assays (ELISAs) revealed IgG antibodies to Dsg1 but not to Dsg3. Complementary DNA (cDNA) transfection method to COS-7 cells and novel ELISAs using eukaryotic recombinant proteins of human Dsc1, Dsc2, and Dsc3 confirmed specific IgG reactivity with Dsc3. (2) A women in her 70s presented with pustular skin lesions on the left fingers with typical histopathological features. Immunoblotting and ELISAs did not detect antibodies to either Dsg1 or Dsg3. Conversely, immunoblotting detected IgG antibodies to Dscs, cDNA transfection method revealed IgG reactivity only with Dsc3, and findings from ELISAs showed that IgG reacted weakly with Dsc2 and strongly with Dsc3. Autoantibodies to Dscs, particularly to Dsc3, may play a pathogenic role in some cases of pemphigus vegetans.

Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells

Genetic modification of human embryonic stem cells (hESCs) using biophysical DNA transfection methods are hampered by the very low single cell survival rate and cloning efficiency of hESCs. Lentiviral gene transfer strategies are widely used to genetically modify hESCs but limited transduction efficiencies in the presence of feeder or stroma cells present problems, particularly if vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped viral particles are applied. Here, we investigated whether the recently described semen derived enhancer of virus infection (SEVI) and alternative viral envelope proteins derived from either Gibbon ape leukaemia virus (GALV) or feline leukaemia virus (RD114) are applicable for transducing hESCs during co-culture with feeder or stroma cells. Our first set of experiments demonstrates that SEVI has no toxic effect on murine or hESCs and that exposure to SEVI does not interfere with the pluripotency-associated phenotype. Focusing on hESCs, we were able to further demonstrate that SEVI increases the transduction efficiencies of GALV and RD114 pseudotyped lentiviral vectors. More importantly, aiming at targeted differentiation of hESCs into functional somatic cell types, GALV pseudotyped lentiviral particles could efficiently and exclusively transduce hESCs grown in co-culture with OP9-GFP stroma cells (which were often used to induce differentiation into haematopoietic derivatives).

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies.

ChemInform Abstract: Development of Tissue Pressure‐Mediated Transfection Method Aimed at Organ‐Specific Gene Expression Control

AbstractReview: 34 refs.

Nanoparticles Deliver Triplex-forming PNAs for Site-specific Genomic Recombination in CD34+ Human Hematopoietic Progenitors

Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34(+) cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the β-globin locus led to levels of site-specific modification in the range of 0.5-1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.

Development of Tissue Pressure-mediated Transfection Method Aimed at Organ-specific Gene Expression Control

A mechanism-based logical approach is a mainstream of current novel drug therapy development in the context of these trends and, therefore, the elucidation of gene function and the molecular level mechanism analysis of diseases at an individual level in mammals are essential in addition to that in cultured cells. In vivo gene transfection techniques are also indispensable for these purposes as well as the evaluation of gene therapy and nucleic acid-based therapy approaches and clinical applications during the process of development of novel drug therapies. Various recombinant virus and synthetic carrier-mediated transfection methods have been reported, however, above all, naked plasmid DNA transfection without virus vectors, synthetic carriers and special physical devices has attracted much attention, because of its advantages including convenience of preparation and handling and lack of toxicity associated with the transfection agents. In this review, I collect the information of these naked plasmid DNA transfection methods involving tissue pressure-mediated transfection from the comprehensive view point including side effects. Additively, the key physiological phenomena affecting transgene expression, especially activation of transcriptional factors, are reviewed. Combined with conventional approach based with biodistribution control, regulation of physiological change in transfected cells will provide spatial- and temporal-controlled transgene expression at various organs, which leads us to elucidate mechanism of diseases and to develop novel drug therapy in near future.

Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR

In animals, microRNAs (miRNAs) bind to the 3' UTRs of their target mRNAs and interfere with translation, although the exact mechanism of inhibition of protein synthesis remains unclear. Functional miRNA-binding sites in the coding regions or 5' UTRs of endogenous mRNAs have not been identified. We studied the effect of introducing miRNA target sites into the 5' UTR of luciferase reporter mRNAs containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a microribonucleoprotein complex would not interfere with the initiation of translation. In human HeLa cells, which express endogenous let-7a miRNA, the translational efficiency of these IRES-containing reporters with 5' let-7 complementary sites from the Caenorhabditis elegans lin-41 3' UTR was repressed. Similarly, the IRES-containing reporters were translationally repressed when human Ago2 was tethered to either the 5' or 3' UTR. Interestingly, the method of DNA transfection affected our ability to observe miRNA-mediated repression. Our results suggest that association with any position on a target mRNA is mechanistically sufficient for a microribonucleoprotein to exert repression of translation at some step downstream of initiation.

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5′ flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules β-galactosidase (β-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express β-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

514. mRNA Transfection Results in Efficient Overexpression of Transgenes in Leukemic Cell Lines and Hematopoietic Stem Cells

DNA-Vector transfection of eukaryotic cells needs transport of the DNA to the nucleus for efficient transcription and subsequent translation. In suspension cells most DNA-transfection methods are either inefficient (e.g. lipofection) or stressful for the cells (electroporation or nucleofection), which leads to high cell losses during the transfection process. We report the use of in vitro transcribed, capped and polyadenylated mRNA for the transfection of leukemic cell lines in comparison to DNA delivery. Templates for in vitro transcription were generated by PCR. The forward primer included the T7-promoter and the reverse primer a fragment of the beta-globin 3'UTR to increase the in vivo half life of the mRNA. Electroporation of GFP-mRNA into Jurkat, K562 or Kg1a lead to a GFP overexpression in 95 % of the transfected cells, with survival rates of 80 to 90%. Electroporation with pEGFP-N1 using the same pulse conditions resulted in GFP-overexpression in 62% of Jurkat or 9% of K562, with survival rates of 50% and 40 %, respectively. Our results suggest that electroporation of DNA is more toxic than mRNA transfection. GFP-expression in mRNA transfected cells was visible two hours after the transfection and peaked at about 12h. In contrast to DNA-transfection, mRNA mediated GFP expression levels were uniform in transfected cells. To investigate whether mRNA transfection results in functional transgene expression, mRNA coding the chemokine receptor CXCR4 was generated and electroporated into CXCR4 negative K562 cells. Flow cytometric analyses showed CXCR4 overexpression in more than 90% of transfected cells. Calcium flux measurements displayed a robust increase of intracellular free calcium in CXCR4-mRNA transfected K562 upon binding of the CXCR4 ligand SDF-1, while no calcium flux was visible in GFP-mRNA transfected or untransfected K562. Electroporation of GFP-mRNA into CD34+ hematopoietic progenitor cells or mesenchymal stem cells resulted in GFP overexpression in > 50% of transfected cells, respectively. Experiments are under way to further improve the mRNA transfection for primary cells. mRNA transfection is a highly efficient means for transient overexpression of transgenes in various cell lines and primary cells.

407 AN ATTEMPT TO GENERATE TRANSGENIC CHICKENS BY INJECTION OF EGFP OR YFP GENE-TRANSFECTED BLASTODERMAL CELLS

Transgenic chickens have been generated from blastodermal chimeric chickens created by an injection of the foreign DNA-transfected blastodermal cells into recipient embryos. However, there has been no method that allows the efficient foreign DNA transfection into chicken primary cells without using viral vectors. This method using viral vectors has a limitation on the size of DNA that can be transfected, and homologous recombination is not possible with this method. This study was conducted to establish a DNA transfection method using electroporation, which can transfect up to 5 kb DNAs into cells. Embryos at stage X were obtained from freshly laid eggs (200) of Barred Plymouth Rock chickens. Blastodermal cells (105 cells) were then collected from them and transfected with the linearized EGFP or YFP gene by electroporation using a Nucleofector Device (Amaxa, Inc., Gaithersburg, MD, USA). After transfection, the transfected chicken blastodermal cells were injected into White Leghorn recipient embryos to produce chimeric chickens. By 48 h after gene transfection, EGFP or YFP gene expression in the cells was easily observed under a fluorescence microscope. The gene expression rate, however, could not be determined, because the cultured chicken blastodermal cells were not able to separate and it was impossible to load them on the flow cytometer to determine the rate. The blastodermal cells transfected with the EGFP or YFP gene were cultured over night and injected into recipient embryos. The manipulated embryos were cultured ex ovo until they hatched. Dead embryos before hatch were dissected and observed under the fluorescence microscope to determine whether they had fluorescent organs. EGFP or YFP gene expression was detected at several areas: head, somite, inner tissues, and limb bud of embryos, but not germinal tissues. Six female chicks and 10 male chicks were obtained. Five females and 6 males were raised until they were sexually mature. Five female chickens were artificially inseminated with semen of Barred Plymouth Rock males to obtain progeny from donor transfected cells. However, no progeny was obtained. Thus the contribution of donor cells to the germ cells could not be determined, and it was not clear if the expression of EGFP gene or YFP gene was stable. Furthermore, semen could not be collected from six males because of a technical problem, and males were not mated. Although a large number of cells must be used to transfect a foreign gene, the novel gene transfer method in chicken in this study has great significance in the field of transgenic chicken production. Even if the gene expression is transient, this transfer method will enable us not only to integrate more than 5 kb DNA into the avian genome but also to induce homologous recombination in cultured avian cells.

P120 catenin translocation is involved in enhancement of hepatoma cellular malignant features

To investigate the relationship between P120ctn translocation and hepatocellular carcinoma cell malignant features and the relationship between p 120ctn and β-catenin translocation in cell signaling. Human hepatocellular carcinoma cells were over expressed with p 120ctn isoform 3A using a DNA transfection method. The effects of transfection and expression of p 120{ctn} and its binding capacity to E-cadherin were examined using immunoprecipitation and immunoblotting methods. p 120ctn subcellular localization and its relation with β-catenin were detected using immunofluorescent microscopy. p 120{ctn} phosphorylation was produced by EGF treatment. Cell adhesion, cell migration and cell proliferation were also examined in this study. We found that p120{ctn} expression was increased after transfection and the binding capacity of P 120{ctn} to E-cadherin was enhanced. Tyrosine phosphorylation of p 120{ctn} increased after transfection and EGF treatment. p 120{ctn} and β-catenin cellular localization displayd a membrane and cytoplasmic expression pattern, but they translocated into the nucleus for relocalization after p 120{ctn} overexpression plus EGF stimulation. Cell adhesion ability was increased and migration ability reduced after transfection without EGF. Following transfection without EGF cellular proliferation was reduced, but increased after EGF treatment. Our results suggest that p 120ctn plays an important role in hepatocellular carcinoma cell adhesion, migration and proliferation. In addition there is a relationship between p 120ctn and β-catenin subcellular localization and signaling.

Reversal of aberrant splicing of beta-thalassaemia allele (IVS-2-654 CT) by antisense RNA expression vector in cultured human erythroid cells

The antisense fragment targeting the aberrant splice sites of the beta-thalassaemia allele, IVS-2-654 C-->T (beta654), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the beta654 mutant pretranscript in cultured beta654 erythroid cells by the lipofectin-mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the beta654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19 (day 0 after transfection) to 0.58 (day 8) in beta654/beta654 homozygous erythroid cells, and from 0.45 (day 0) to 0.83 (day 8) in beta654/betaA heterozygous erythroid cells, as determined by the ratio of normally spliced beta-globin transcript over total beta-globin transcript. Correspondingly, the ratios of globin chain biosynthesis (beta/alpha) increased from 0.16 (day 0) to 0.52 (day 8) in beta654/beta654 erythroid cells, and from 0.39 (day 0) to 0.84 (day 8) in beta654/betaA erythroid cells. Antisense RNA had no significant effect on the splicing pattern in betaA/betaA erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence). In addition, we did not observe side-effects on cytological features after the introduction of pCMVA. All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of beta654 mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.