Showing papers on "Marker gene published in 1993"

Rapid Production of Multiple Independent Lines of Fertile Transgenic Wheat (Triticum aestivum)

Abstract: Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding [beta]-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred.

Efficient gene transfer into myocardium by direct injection of adenovirus vectors.

Abstract: Previous studies have established that gene transfer into myocardial cells in vivo is detectable after direct injection of plasmid DNA. Recently, adenovirus vectors have been shown to provide an efficient method for gene transfer into a wide range of tissues. Therefore, this study sought to assess the efficiency and stability of adenovirus-mediated gene transfer into myocardium and to compare this method with that using plasmid-based gene transfer techniques. Adult rats underwent myocardial injection via a subdiaphragmatic approach. Gene transfer efficiency was compared using direct injection of an adenovirus vector encoding for the marker gene beta-galactosidase (beta-gal), a control adenovirus vector encoding for the cystic fibrosis transmembrane conductance regulator gene, a plasmid encoding for beta-gal, or a control plasmid. Hearts infected with an adenovirus vector containing the beta-gal gene showed significantly increased beta-gal enzymatic activity compared with hearts injected with beta-gal plasmid. Histological examination revealed that cardiac myocytes were the target of adenovirus-mediated gene transfer. A time course of gene expression showed that beta-gal enzymatic activity peaked during the first week following injection. Adenovirus vectors provide an efficient but transient method for in vivo gene expression in myocardium.

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Abstract: Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.

Efficient and selective adenovirus-mediated gene transfer into vascular neointima.

Abstract: BACKGROUNDPrevious attempts to target arterial smooth muscle cells (SMCs) for gene delivery using liposomal or retroviral methods were limited by low transfection efficiency. We therefore evaluated the efficiency of adenovirus-mediated gene delivery in cultured vascular SMCs and in an in vivo model of balloon injury-induced SMC cell proliferation.METHODS AND RESULTSWe used a recombinant adenovirus, Ad.RSV beta gal, which contained the beta-galactosidase (beta-gal) histochemical marker gene. For in vitro studies, rat aortic SMCs were incubated in media containing Ad.RSV beta gal for 5 to 120 minutes. The proportion of SMCs expressing the beta-gal gene product increased from 25% (5-minute exposure) to 80% (120-minute exposure). For in vivo studies, uninjured and injured rat carotid segments were incubated with 0.5 to 1.0 x 10(9) pfu Ad.RSV beta gal for 45 minutes. Uninjured arteries showed adenovirus-mediated gene transfer limited to the endothelium. Injured arteries were exposed to adenovirus 0, 3, 7, or 1...

Creation of mice expressing human antibody light chains by introduction of a yeast artificial chromosome containing the core region of the human immunoglobulin kappa locus.

Abstract: We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.

Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S locus glycoprotein gene promoter.

Abstract: The promoter of the S Locus Glycoprotein (SLG) gene of Brassica is a tightly regulated promoter that is active specifically in reproductive organs. In transgenic tobacco, this promoter is active exclusively in cells of the pistil and in pollen. We transformed tobacco with truncated versions of the SLG13 promoter fused to the beta-glucuronidase reporter gene. We show that the promoter has a modular organization and consists of separable DNA elements that independently specify pistil- and pollen-specific expression. A 196-bp region (-339 to -143) is sufficient to confer stigma and style specificity to the marker gene. Two distinct, but functionally redundant, domains (-415 to -291 and -117 to -8) allow specific expression of the gene in pollen. The functional domains identified within the SLG13 promoter contain sequence elements that are highly conserved in different alleles of the SLG gene and in the S Locus Related SLR1 gene.

Detection of quantitative trait loci from frequency changes of marker alleles under selection

Abstract: Summary A method was developed to estimate effects of quantitative trait loci (QTL) by maximum likelihood using information from changes of gene frequency at marker loci under selection, assuming an additive model of complete linkage between markers and QTL. The method was applied to data from 16 molecular and coat colour marker loci in mouse lines derived from the F2 of two inbred strains which were divergently selected on 6-week weight for 21 generations. In 4 regions of the genome, marker allele frequencies were more extreme than could be explained by sampling, implying selection at nearby QTL. An effect of about 0-5 standard deviations was located on chromosome 11, and accounted for nearly 10% of the genetic variance in the base population. QTL with effects as small as 0-2 phenotypic standard deviations could be detected. For typing of a given number of individuals, the power of detection of QTL is very high compared to, for example, analysis of an F2 population. The joint effects of linkage and selection were investigated by Monte Carlo simulation. Marker gene frequencies change little as a consequence of selection at a QTL unless the marker and QTL are less than about 20 cM apart.

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with gibbon ape leukemia virus envelopes

Abstract: With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.

Targeted replacement of a gene without endogenous and selectable residual sequences

Abstract: A process is disclosed for replacing a gene or a gene segment in the germ line of a non-human mammal by a homologous gene or by a homologous gene segment of another mammal The process comprises the following steps: (i) an embryonic parent stem line is transfected by a selectively marked recombination vehicle; (ii) stably transfected cell clones are selected on the basis of the presence of the marker gene; (iii) these are selected in a targeted way by PCR and/or Southern Blot; (iv) these are injected into the blastocysts of the non-human mammal; and (v) the blastocysts are transferred into surrogate mothers The process is characterized in that during recombination the endogenous gene or the endogenous gene segment is functionally replaced in a single step by the homologous gene or by the homologous gene segment thanks to the selectively marked recombination vehicle

A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots

Abstract: There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an introncontaining GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.

A rapid screening method to detect nonsense and frameshift mutations: identification of disease-causing APC alleles.

Abstract: A functional screen for nonsense and frameshift mutations has been devised that allows genes of interest to be scanned in segments. This assay is based on the cloning of these segments in-frame with a colorimetric marker gene ( lacZ ) followed by screening for the level of functional activity from the marker polypeptide ( β -galactosidase). Individuals at risk for any one of a number of genetic diseases, in particular familial adenomatous polyposis coli (APC), can be quickly screened for chain-terminating mutations introduced by stops and frameshifts. At present, scanning of the APC gene for mutation requires significant effort because it is a large gene and most APC mutations are unique. Therefore, this assay offers a powerful option for the diagnosis of this and other genetic diseases, as well as great potential for the development of a similar rapid screen to detect APC mutations in colorectal adenomas and carcinomas.

Selectable Marker Genes Engineered for Specific Expression in Target Cells for Plant Transformation

Abstract: A wound-induced promoter (AoPRl) isolated from Asparagus officinalis was shown by GUS reporter gene analysis to be active during callus formation in tissue cultured leaves from transgenic tobacco plants Unlike other promoters commonly used to drive expression of marker genes during transformation (eg Nos, MAS-35S and CaMV35S), the AoPR1 promoter showed strong expression at wound sites during tobacco leaf disk transformation but was expressed at extremely low levels in the leaves, roots and seeds of the mature plant A plant transformation vector was constructed in which nptII expression was placed under the control of the AoPR1 promoter This construct was then used in transformation experiments which resulted in the production of a large number of transgenic tobacco and potato plants In leaves, roots and tubers (in the case of potato) of these plants, the marker gene protein product (NPTII) was present in low amounts compared to the levels found in control transgenic plants produced using a CaMV35S-nptII gene as a selectable marker

Electroporation of rapeseed protoplasts - transient and stable transformation.

Abstract: Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10-5 and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.

An Isozyme Marker for Pollen Fertility Restoration in the Pampa cms System of Rye (Secale cereale L.)

Abstract: Hybrid breeding in rye (Secale cereale L.) based on cytoplasmic male sterility requires a sufficient restoration of pollen fertility to guarantee full seed set in the hybrid variety. Therefore the selection of effective restorer lines is an important goal in the breeding process. An F2 generation tracing back to a cross of a nonrestorer line as seed parent and a restorer line, and subsequent three-way crosses between the corresponding F1 plants and a nonrestorer line were used to find marker genes for pollen fertility restoration. A linkage between the Prx.7 isozyme locus on chromosome 1R and a putative major gene (or gene complex) for pollen fertility restoration with a recombination value of about 20 % was found. To our knowledge, this is the first case of a marker gene for this trait in rye.

Screening assay for the identification of immunosuppressive drugs

Abstract: A method for identifying compounds capable of inducing immunosuppression by inhibiting the CD28 signal transduction pathway and T cell comprises exposing cultured T cells to one or more test compounds. The T cells are obtained from a T cell line which stably incorporates DNA sequence comprising in reading frame an enhancer region responsive to a CD 28-regulated nuclear binding protein and a marker gene. The cells are cultured under conditions which result in activation of both the T cell receptor and the CD28 receptor, resulting in enhanced expression of the marker gene. Test compounds which inhibit expression of the marker gene are considered as candidates for immunosuppressive drugs.

Estimation of genetic parameters using linkage between a marker gene and a locus underlying a quantitative character in F2 populations

Abstract: Estimation of genetic parameters using linkage between a marker gene and a locus underlying a quantitative character in F 2 populations

Screening assay for the identification ov novel immunosuppressives using cultured T cells

Abstract: A method for identifying compounds capable of inducing immunosuppression by inhibiting the CD28 signal transduction pathway and T cell comprises exposing cultured T cells to one or more test compounds. The T cells are obtained from a T cell line which stably incorporates DNA sequence comprising in reading frame an enhancer region responsive to a CD28-regulated nuclear binding protein and a marker gene. The cells are cultured under conditions which result in activation of both the T cell receptor and the CD28 receptor, resulting in enhanced expression of the marker gene. Test compounds which inhibit expression of the marker gene are considered as candidates for immunosuppressive drugs.

Improving transformation method for industrial yeasts: construction of adh1-apt2 gene and using electroporation

Abstract: Strong expression system of yeast dominant selection marker gene was constructed for transformation of industrial yeasts. Bacterial aminoglycoside phosphotransferase II (APT2) gene from Escherichia coli transposon Tn903 was inserted between a yeast alcohol dehydrogenase I (ADH1) gene promoter and cytochrome c1 (CYC1) gene terminator. This ADH1-APT2 gene was strongly expressed in the transformed yeasts even in the case of integration type plasmid. Using this strong expressive dominant selection marker gene, five strains in nine industrial yeasts which were tested could not be transformed by lithium acetate method, but these industrial yeasts could be transformed by electroporation.

Insertion mutants of the vaccinia virus. The effect of inactivating E7R and D8L genes on the biological properties of the virus

Abstract: The integrative plasmids with the expressive marker gene for beta-galactosidase were constructed for insertional inactivation of nonessential genes E7R and D8L of vaccinia virus located in the central region of the viral genome. Inactivation of the D8L gene in the strains WR and LIVP results in smaller viral plaques in the culture of chicken embryo cells, decreases the viral ability to propagate in mouse brain and has no effect on the size and character of damage in intracutaneous infection of rabbits. Inactivation of E7R gene did not affect the known biological properties of the virus. The existence of the nonessential genes in the central region of the viral genome, inactivation of which does not result in viral attenuation, can be used for increase of antigenic activity of the live attenuated vaccines.

Molecular genetics in Streptococcus thermophilus: from transformation to gene expression

Abstract: 5ummary Streptococcus salivarius subsp thermophilus (S thermophilus) is a homofermentative, thermophilic lactic acid bacteria, used in dairy starter cultures. Despite its widespread and long-term use, its molecular biology and genetics have only recently started to be investigated. We report here the isolation and characterization of a cryptic, 3-kb plasmid which was converted into an E coli S thermophilus shuttle vector. Using this and other plasmids, transformation was optimized and used to integrate non-replicative plasmids into the bacterlal genome. Resulting cointegrates were able to amplity. Resolution of the cointegrates was used for gene-replacement by introducing an in vitro generated deletion into a genomic located structural gene. By the same mechanism, a heterologous, promoter-Iess marker gene was inserted ante the genome between the permease and ~galactosidase gene of the lactose operon. It was thereafter expressed as a functional part of the operon and followed lactose regulation. The control region of the lactose operon was investigated by analyzing promoter up and down mutants.

Lipid hydroperoxide-resistance gene in Saccharomyces cerevisiae : utilization as a selectable marker gene for yeast transformation

Abstract: Two kinds of DNA fragments concerned with resistance against lipid hydroperoxide were cloned from the yeast Saccharomyces cerevisiae. When the recombinant plasmid carrying the DNA fragment was introduced into the cells of S. cerevisiae which do not have an appropriate auxotrophic marker, the transformants could be selected by their resistance to t-butyl hydroperoxide. By Southern hybridization analysis, such transformants were found to contain the recombinant plasmid, suggesting that the DNA fragment could be used as a selection marker for the transformation of S. cerevisiae.

A quantitative assay for trans-activation by HIV-1 Tat, using liposome-mediated DNA uptake and a parallel ELISA system.

Abstract: A cellular assay is described in which transient high-level expression of a heterologous reporter gene (chloramphenicol acetyltransferase, CAT) driven by the HIV LTR is used to determine trans-activation in a cell line constitutively expressing Tat. The use of a parallel ELISA system to determine effects on expression of CAT and of the neomycin phosphotransferase (NPT) marker gene effectively eliminated sample variability caused by cumulative processing errors or cell culture conditions. In addition the use of cationic liposome-mediated transfection minimized delay between DNA treatment that initiates trans-activation and addition of inhibitors, thereby eliminating background expression levels in treated samples. The assay has the potential to discriminate between inhibition of trans-activation and nonspecific effects such as inhibition of transfection and cytotoxicity. It has been adapted to a 96-well format suitable for high-throughput screening of natural products and synthetic chemicals.

Homogenotization of gene-targeting events

Abstract: Homozygotic cells are obtained by employing homologous recombination with a construct comprising a marker gene. The marker gene allows for selection without amplification and by employing elevated levels of the antibiotic to which the marker gene imparts resistance, gene conversion can occur, where in a diploid host, both copies of the target locus will be the same. In this manner, knock-outs of genes can be readily achieved without requiring two steps of homologous recombination.