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Results for “expression efficiency”

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At least 505 records · Page 28Linked to original sources
PubMed1987

Characterization of trans-acting factor(s) regulating beta-globin gene expression by in vivo competition.

A beta-globin/TK fusion gene was microinjected into non-erythroid cells (Ltk- cells) and erythroid cells (murine erythroleukemia (MEL) cells), and the interactions of the regulatory cellular factors with the beta-globin sequences were investigated by the in vivo competition experiment. The fusion gene was expressed efficiently in Ltk- cells. This expression was inhibited by a co-injection with a three-fold molar excess of the 5'-flanking sequence of the beta-globin gene or with a nine-fold molar excess of the mammary tumor virus LTR, but not with the alpha-globin gene. The fusion gene was expressed very poorly in the uninduced MEL cells and highly in the induced MEL cells. The co-injection of the beta-globin gene did not affect expression in the MEL cells in either uninduced or induced conditions.

Animals↗
PubMed1990

A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria.

A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed. The expression vectors contain various promoters (pNm, plac, ptac and pS1) for expression of foreign genes. The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by beta-galactosidase activity measurements. Of the promoters assayed in E. coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the NmR promoter. The GmR gene, serving as a selectable marker for the plasmids, was efficiently expressed in R. meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, pGm was also used to construct an expression vector. The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments. All lac fusion vectors contain the E. coli lacZ gene or the complete lac operon facilitating quantification of expression.

Base Sequence↗
PubMed1992

A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa.

A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aeruginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUV5/lacIq-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This element, designated miniD-180, was stably integrated into the P. aeruginosa PAO1 chromosome. The second component of this system includes several improved broad-host-range expression vectors containing the T7 gene 10 promoter and multiple cloning site (MCS). These vectors (pEB8, pEB11, and pEB12) contain transcriptional terminators (T1(4)) upstream from the T7 promoter, and T7 terminators downstream from the MCS. Because the T7 promoter is somewhat leaky in these vectors, pEB14 was constructed to decrease transcription of target genes by basal levels of T7 RNA polymerase. This vector contains a core sequence of the lac operator located 19 bp downstream from the transcriptional start point of the T7 promoter, thereby providing a dually regulated system. The utility of this system was demonstrated by placing a promoterless chloramphenicol acetyltransferase (CAT) cassette under control of the T7 promoter and monitoring the isopropyl-beta-D-thiogalactopyranoside-dependent accumulation of CAT in cell-free extracts of P. aeruginosa. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction, at which time this polypeptide represented up to 20% of the total soluble protein.

Chloramphenicol O-Acetyltransferase↗
PubMed2004

Increased protein expression from adenoviral shuttle plasmids and vectors by insertion of a small chimeric intron sequence.

Adenoviruses are widely used as gene transfer vehicles because they can be produced at high titers, they have a large transgene capacity, and can transduce both dividing and non-dividing cells. One disadvantage of adenoviral vectors is the narrow therapeutic window due to a dose-dependent humoral as well as a T-cell dependent host immune response directed against the transduced cells, that leads to a reduction of transgene expression with time. By increasing the levels of protein expression from transcription units, vector titres may be decreased without a significant loss of transgene expression. Introns are required for efficient expression of many protein coding genes. In addition, splicing signals are required for some genes in order to be translated. Therefore, a chimeric intron sequence was introduced at the 3' end of the transgenes to study its effect on protein expression from adenoviral vector constructs. Transfection of 293 cells with the adenoviral shuttle plasmids pMH4-EGFP-Int, pMH4-E314.7-Int, pMH4-BclX(L)-Int and pMH4-Luc-Int lead to a 1.8-20-fold increase in protein expression as compared to constructs lacking an intron. Injection of Ad-CMV-Luc-Int into the brain of C57Bl/6 mice results in an approximately three-fold increase of luciferase activity as compared to Ad-CMV-Luc. In conclusion, insertion of an intron sequence leads to a significant increase in transgene expression both in vitro and in vivo, thus allowing for reduction of the adenoviral vector dose used.

Adenoviridae↗
PubMed1999

The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability.

The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation C134S was purified to homogeneity but appeared to be very unstable. In contrast, the C167S mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the C167S BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease.

Amino Acid Substitution↗
PubMed2001

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.

Base Pair Mismatch↗
PubMed1994

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA.

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.

Base Sequence↗
PubMed2005

Stimuli-responsive polymers in gene delivery.

Recent interest in clinical therapy has been directed to deliver nucleic acids (DNA, RNA or short-chain oligonucleotides) that alter gene expression within a specific cell population, thereby manipulating cellular processes and responses, which in turn stimulate immune responses or tissue regeneration, or blocks expression at the level of transcription or translation for treatment of several diseases. Both ex vivo and in vivo gene delivery can be achieved mostly by using a delivery system (vector). Viral vectors exhibit high gene expression, but also have very significant side effects. Mainly cationic polymeric systems are used as nonviral vectors, although usually with low levels of transfection. Through the use of stimuli-responsive polymers as novel vectors for gene delivery, two benefits can be obtained: high gene expression efficiency and more selective gene expression.

Gene Expression↗
PubMedDate not supplied

Calretinin promoter for suicide gene expression in malignant mesothelioma.

To achieve specific and efficient expression of transfected suicide gene in malignant mesothelioma cells, we explored the 5-flanking region (2.2 kb) of the calretinin gene as a tumor-specific promoter. RT-PCR analysis demonstrated the expression of calretinin mRNA in two human malignant mesothelioma cell lines (H2052 and 211 H). A reporting vector containing the calretinin promoter (pCAL-LUC) exhibited relatively high luciferase activity in these cell lines. After transfecting an expression vector containing the calretinin promoter-bound thymidine kinase gene (pCAL-TK) into the cells, we examined their sensitivity to ganciclovir (GCV). In the mesothelioma cell lines, the pCAL-TK transfected cells became about 100 times more sensitive to GCV than the parental cells. The calretinin promoter seems to be a promising candidate as a specific and efficient promoter in malignant mesothelioma.

Antiviral Agents↗
PubMed1995

Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase.

Triple tandemly repeated sequences and the corresponding complementary sequence are known to exist in the 5'-terminal regulatory region of the human gene for thymidylate synthase (TS). To examine the function of these sequences, a set of deletion mutants was prepared and used in a transient expression assay. The results showed that at least one repeated sequence and its complementary sequence were necessary for the efficient expression of the gene. As another approach to understanding the function of this unique structure, DNA polymorphism in the same region was analyzed. In addition to the TS gene with the triple tandem repeat, the TS gene with a double tandem repeat was found in genomes of normal human subjects at an estimated frequency of 19% when genomes of 21 unrelated Japanese were analyzed. The expression activity of a reporter gene linked to the promoter region of the human TS genes with the two types of repeated sequence was examined and the result showed that the expression activity of the gene with the double repeat was lower than that of the gene with the triple repeat in the transient expression assay. Thus, it appears that the unique repeated sequences in the 5'-terminal region of the human TS gene are polymorphic and contribute to the efficiency of expression of the gene.

Base Sequence↗
PubMed2000

[Retroviral-mediated high efficient in vitro expression of human coagulation factor VIII].

OBJECTIVE: To develop a retroviral-mediated high efficient expression system of human coagulation factor VIII. METHODS: The retroviral vector LNC-VIIIBD was generated by cloning a B-domain-deleted FVIII cDNA (760aa - 1639aa) into retroviral vector pLNCX. Several cell lines including NIH3T3, CHO, COS-7 and human hepatic cell line L-02 were infected with viral supernatant from the highest productive PA317 clones. The antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of F VIII BD mRNA. RESULTS: Human FVIII was expressed in all four target cells. The highest expression was observed in NIH3T3, the procoagulant activity of secreted FVIII was up to 1.6 U, and the FVIII antigen was 500 ng by 10(6) cells/ml in 24 hours, respectively. CONCLUSION: The constructed retroviral vector was able to generate high level expression of human FVIII in some cell lines, and it might have potential utility in the gene therapy for Hemophilia A.

3T3 Cells↗
PubMed1989

Expression of a viral gene in insulin-producing cell lines renders them susceptible to immunological destruction.

The gene coding for the glycoprotein D of herpes simplex virus type 1 was cloned into plasmids under the transcriptional control of the SV40 promoter-enhancer or the rat insulin 1 promoter-enhancer sequences. These plasmids were transfected into rat insulinoma cells (RINm5F) and mouse NIH/3T3 cells and the expression of glycoprotein D was examined using cell surface immunofluoresence. The rat insulin 1 promoter-enhancer sequences directed efficient expression in RINm5F cells, but not in NIH/3T3 cells. In contrast, the SV40 promoter-enhancer sequences worked well in NIH/3T3 cells, but not in RINm5F cells. Expression of glycoprotein D did not interfere with insulin production by RINm5F cells. When stable cel lines expressing glycoprotein D were exposed to anti-herpes simplex virus type 1 antibodies and complement, they were destroyed. These studies provide additional evidence that specific promoter-enhancer elements are required for efficient gene expression in certain cell types and demonstrate that the expression of foreign antigens on the surface of insulin-producing cells can lead to their immunological destruction.

Animals↗
PubMed2005

Efficient gene transfer into mouse embryonic stem cells with adenovirus vectors.

Efficient and transient gene transfer into embryonic stem (ES) cells is expected to be of use for basic studies in developmental biology and for applications in regenerative medicine. Here, we report the development of an adenovirus (Ad) vector that efficiently expresses foreign genes in mouse ES (mES) cells. We prepared four LacZ-expressing Ad vectors, each of which contained one of the following: Rous sarcoma virus (RSV), cytomegalovirus (CMV), beta-actin promoter/CMV enhancer (CA), or EF-1alpha promoter. While the RSV and CMV promoters were inactive in mES cells, the CA and EF-1alpha promoters strongly drove LacZ expression in more than 90% of the mES cells. The EF-1alpha promoter was found to be slightly more efficient than the CA promoter. mES cells were found to express the Ad primary receptor, coxsackievirus and adenovirus receptor, suggesting that while Ad vectors could introduce the exogenous gene into mES cells, the choice of a suitable promoter was critical for efficient gene expression. Fiber-mutant Ad vectors containing RGD or polylysine peptide on the fiber knob mediated efficient LacZ expression, not only in mES cells, but also in feeder cells. Exogenous expression of Oct-3/4 or the dominant-negative mutant of STAT3 (STAT3F) by conventional Ad vectors containing the EF-1alpha promoter promoted the differentiation of mES cells into the cells of three germ layers, and STAT3F-mediated differentiation was rescued by the coexpression of Nanog. These results suggest that Ad vectors can be used for basic research using ES cells and that they may be of great utility for therapeutic applications in gene-modified regenerative medicine based on ES cells.

Actins↗
PubMed2001

Receptor-mediated gene delivery to human peripheral blood mononuclear cells using anti-CD3 antibody coupled to polyethylenimine.

Gene transfer to primary cells, especially to lymphoid cells, using a nonviral delivery system has been very challenging. In the present studies, we have used a cationic polymer, polyethylenimine (PEI) coupled to an anti-CD3 antibody for achieving receptor-mediated gene delivery to human peripheral blood mononuclear cells (PBMC). Naive, unstimulated PBMC did not express transfected genes, whereas the transgenes were expressed efficiently in PHA activated PBMC. Transiently expressed gene products were detected maximally at 24 and 48 h following transfection. Gene expression was detected until 96 h with a gradual diminution in the signal after 48 h. Receptor-mediated gene delivery was successfully used for freshly isolated, as well as previously frozen lymphocyte samples. The transfections performed using ligands other than anti-CD3 were not as efficient as anti-CD3-PEI. These results suggest that in addition to receptor-mediated endocytosis, signaling subsequent to engagement of the CD3 receptor with anti-CD3-PEI appears to be important for the efficacy of anti-CD3-PEI mediated gene delivery.

CD3 Complex↗
PubMed2002

Gene transduction efficiency in cells of different species by HIV and EIAV vectors.

The ability of human immunodeficiency virus (HIV)- and equine infectious anaemia virus (EIAV)-based vectors to transduce cell lines from a range of species was compared. Both vectors carried the vesicular stomatitis virus G (VSV-G) envelope protein and encoded an enhanced green fluorescent protein (eGFP) gene driven by a human cytomegalovirus (CMV) early promoter. Immunostaining for viral core proteins and VSV-G was used to demonstrate that the HIV and EIAV vector preparations contained similar numbers of virus particles. Various cell lines were transduced with these vectors and the transduction efficiency was estimated by measuring eGFP expression. Efficient transduction by both vectors was observed in human, hamster, pig, horse, cat and dog cell lines, although EIAV vector was about 10-fold less efficient in human, hamster and pig cells normalised to the total number of viral particles. This could be partly explained by the lower RNA genome levels per particle for EIAV as measured by real-time RT-PCR. Rodent cells appeared to be transduced inefficiently with both vectors, but when the CMV promoter was substituted with the EF1alpha promoter in the HIV vectors, the expression level increased leading to an increase in the measurable level of transduction.

Animals↗
PubMed1994

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.

Antigens, Plant↗
PubMed1997

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription.

Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.

Animals↗
PubMed2001

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.

We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.

Cephalosporins↗