Negative Selection Marker Research Articles

Escherichia coli gpt as a positive and negative selectable marker in embryonal stem cells

Transfection of HPRT − L fibroblasts with a plasmid containing two linked selectable markers genes, gpt and neo, regulated by the same eukaryotic control elements, yielded a 6-fold higher transfection frequency on selection for neo than for gpt. Transfection of HPRT − embryonal stem (ES) cells with the same plasmid yielded high levels of transfectants when selected for neo expression with G418, but a level of transfection greater than two orders of magnitude lower was observed when HAT supplemented medium was used to select for gpt expression. Selection for gpt expression in ES cells with medium containing mycophenolic acid and xanthine gave slightly higher frequencies of transfection, but still considerably lower than that for neo selection. In addition, mycophenolic acid exhibited a general cytotoxicity to ES cells with the window between toxicity of this compound to gpt − ES cells and gpt + ES cells being very narrow. Cells selected with mycophenolic acid and xanthine for expression of gpt remained sensitive to HAT selection. Expression of gpt in a representative ES cell line, selected on mycophenolic acid and xanthine, was verified by Northern analysis and sensitivity to 6-thioguanine. While the level of mRNA expression in this ES cell line was insufficient to support growth via purine salvage when exposed to HAT medium, identical levels of gpt expression in HPRT − L cells, as judged by Northern analysis, allowed for normal growth in HAT medium. This suggests that ES cells place a greater demand on purine nucleotide biosynthesis than L cells. These results are discussed in terms of the use of gpt as a positive and negative selectable marker for gene targeting via homologous recombination in ES cells.

Gene Targeting Constructs: Effects of Vector Topology on Co-expression Efficiency of Positive and Negative Selectable Marker Genes

Targeting of DNA into specific chromosomal loci often involves the use of a negative selectable marker gene to enrich for cell clones that have undergone homologous recombination. In this study we asked if the arrangement of the positive and negative markers in ′knockout′ constructs can influence the expression of a lacZ gene that was used as a negative marker. We show that constructs which differ only in their topology have vastly different co-expression efficiencies. The site of DNA linearization was critical. While linearizing at the 5′-end of the lacZ gene was compatible with efficient lacZ expression, linearizing at the 3′-end was always detrimental. We also demonstrate that the topology of the template was more important than the promoter used to drive lacZ expression.

Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.

A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation.

The Herpes Simplex Virus Thymidine Kinase Gene as a Conditional Negative-Selection Marker Gene in Arabidopsis thaliana

The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures. There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium. Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments. Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control. This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.

The Anti-nptII Gene (A Potential Negative Selectable Marker for Plants).

An efficient negative selection procedure is crucial to the isolation of rare homologous recombinants in gene targeting. Although gene targeting is a common practice in lower eukaryotes and is becoming routine in mammals, its application to plants has not been achieved. In this report, we have evaluated an antisense construct against the neomycin phosphotransferase gene (nptII) as a negative selectable marker. The anti-nptII gene construct was able to suppress nptII expression both transiently and in transformed tobacco (Nicotiana tabacum) calli. A construct was made which includes both a hygromycin-resistance gene and the sense plus antisense genes for neomycin phosphotransferase. Hygromy-cin-resistant calli were obtained after Agrobacterium-mediated transformation. Subsequently, hygromycin-resistant calli were tested for kanamycin sensitivity. The growth on kanamycin medium of calli harboring both the sense and antisense gene constructs was retarded, whereas that of control calli transformed with only the sense nptII gene was not inhibited. Southern blot analysis confirmed the presence of both nptII and anti-nptII genes. Northern blot analyses revealed that antisense transcripts of the nptII gene were made and that the level of sense transcripts was greatly reduced in transgenic calli. These results suggest that the anti-nptII gene could potentially be used as a negative selectable marker for gene targeting in plants.

Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene ( neo R) which is driven by a modified thymidine kinase ( tk) promoter for positive selection. Flanking neo R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.

Patent Evaluation: Bifunctional Selectable Fusion Genes

SummaryNovelty: A bifunctional fusion protein and the sequence encoding it are disclosed. The protein is said to provide both a positive selectable marker from one of the components and a dominant negative selectable marker from the other part. This enables cells carrying the gene to be selected for in the first stages of gene therapy and then allows those cells to be selectively killed when the gene therapy is over.Biology: The bifunctional protein was formed by the fusion of the gene for hygromycin B resistance (positive selectable phenotype) and the HSV-TK gene for ganciclovir sensitivity (negative selectable phenotype). Having both activities expressed as a fusion protein appears to avoid problems of differential expression or loss of one of the genes. The 76 kD fusion protein was expressed in rat-2 cells using a retroviral vector. This allowed both cell selection and then complete cell killing.

Thymidine kinase as a negative selectable marker in Leishmania major

Thymidine kinase as a negative selectable marker in Leishmania major

A dominant positive and negative selectable gene for use in mammalian cells.

We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells.