Mapping of the human CP49 gene and identification of an intragenic polymorphic marker to allow genetic linkage analysis in autosomal dominant congenital cataract

The CP49 protein is an intermediate filament protein expressed specifically in the lens fibre cells of the lens, where it is an important cytoplasmic structural component. Dominant-negative mutations in other intermediate filament proteins, such as keratins, cause disorders characterised by dense cytoplasmic aggregates in specific cell types. The CP49 gene is therefore a good candidate for dominantly inherited forms of cataract. To allow genetic linkage analysis of families with autosomal dominant cataract with respect to CP49, a highly polymorphic intragenic microsatellite marker for this gene has been developed. In addition, both low and high resolution radiation hybrid mapping of the CP49 gene has been completed, placing it very close to microsatellite marker D3S1290 on human chromosome 3q. Furthermore, using the intragenic CP49 microsatellite, linkage was excluded in four families with genetically uncharacterized forms of autosomal dominant congenital cataract.     

Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida.

In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE-ColA protein was expressed in E. coli under conditions of phosphate starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.     

Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance.

The gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger has been cloned next to the SV40 early promoter in a mammalian cells-Escherichia coli shuttle vector. When this construction was introduced into VERO cells it expressed the relevant enzymic activity. Moreover, the puromycin N-acetyl transferase gene has been used as a dominant marker for the selection of transformed mammalian cells able to grow in the presence of the antibiotic.     

Selective Disruption of Genes Transiently Induced in Differentiating Mouse Embryonic Stem Cells by Using Gene Trap Mutagenesis and Site-Specific Recombination

A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development. Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of β-galactosidase (β-Gal). As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected. Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo. Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of β-Gal expression.     

Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F₁ generation.     

Identification of gene expression profile of neural crest-derived cells isolated from submandibular glands of adult mice

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity.     

The UPS locus encoding uroporphyrinogen I synthase is located on human chromosome 11

The expression of the UPS locus encoding uroporphyrinogen I synthase has been investigated in human/mouse somatic cell hybrids. Human and mouse uroporphyrinogen I synthase can be readily distinguished by their isoelectric points. In hybrid cells, both human and mouse isozymes are detected. The multiple human uroporphyrinogen I synthase isozymes segregate as a single unit, as expected if they are the products of a single locus. The absence of new heteropolymers in hybrid cells supports the biochemical evidence that the active enzyme is a monomer. The presence of human uroporphyrinogen I synthase in hybrid clones was correlated with the presence of human chromosome 11, or its enzymatic marker, without exception in 44 independent hybrid lines. All other chromosomes could be eliminated as possible locations for this locus, due to their independent segregation. This report represents the first gene assignment for an enzyme in the heme biosynthesis pathway.     

Limited lifespan in somatic cell hybrids and cybrids

The transformed phenotype is believed to be dominant in fusions between limited lifespan cells and transformed cells, based on heterokaryon experiments and on the isolation of transformed hybrids from mass cultures of fused cells. A series of fusions has been performed between limited lifespan Lesch-Nyhan fibroblast cells and a permanent HeLa cell line with a complementary genetic marker. The growth of independently isolated hybrid clones was followed in parallel with Lesch-Nyhan cells. In fusions involving Lesch-Nyhan cells which had completed about 50% of their lifespan, all hybrids initially show fibroblastic properties. Thirty-five hybrids had a limited lifespan slightly longer than Lesch-Nyhan controls. Three other hybrid clones, and all mass cultures of hybrids, showed the appearance of transformed colonies at a rate of approx. one transformant in 2 × 10₅ hybrid cells. These transformed cells showed anchorage independence, low serum requirement, chromosome loss, and have been maintained in culture for 50–100 population doublings with no signs of senescence. Fusions involving enucleated HeLa cells did not show transformation. Fusions with senescent Lesch-Nyhan cells yielded hybrids which grew beyond the normal Lesch-Nyhan cell lifespan, but which again showed limited lifespan and low frequency transformation. It is concluded that limited lifespan is expressed in a dominant manner in these fusions, and that transformation or “escape from senescence” is a low frequency event requiring the presence of the HeLa nucleus.     

A dominant selectable marker for the construction of recombinant poxviruses

Mycophenolic acid has been shown to be a potent inhibitor of vaccinia virus growth. By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome. When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes. Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.     

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.     

Heterogeneity of Ovarian Theca and Interstitial Gland Cells in Mice

It has been established that two developmentally and functionally distinct cell types emerge within the mammalian testis and adrenal gland throughout life. Fetal and adult types of steroidogenic cells (i.e., testicular Leydig cells and adrenocortical cells) develop in the prenatal and postnatal period, respectively. Although the ovary synthesizes steroids postnatally, the presence of fetal-type steroidogenic cells has not been described. We had previously established transgenic mouse lines in which fetal Leydig cells were labeled with an EGFP reporter gene by the FLE (fetal Leydig enhancer) of the Ad4BP/SF-1 (Nr5a1) gene. In the present study, we examined the reporter gene expression in females and found that the reporter gene is turned on in postnatal ovaries. A comparison of the expressions of the EGFP and marker genes revealed that EGFP is expressed in not all but rather a proportion of steroidogenic theca and in interstitial gland cells in the ovary. This finding was further supported by experiments using BAC transgenic mice in which reporter gene expression recapitulated endogenous Ad4BP/SF-1 gene expression. In conclusion, our observations from this study strongly suggest that ovarian theca and interstitial gland cells in mice consist of at least two cell types.     

Chimeric genes as dominant selectable markers in plant cells

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx^R) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx^R gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.     

A new immunochemical method for the quantitative measurement of specific gene products in man-rodent somatic cell hybrids

Summary An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance a-galactosidase and N-acetyl-a-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-a-galactosaminidase (also known as a-galactosidase B) to hydrolyze a-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.     

A dominant nuclear streptomycin resistance marker for plant cell transformation

Summary Plant cells in photoheterotrophic culture respond to streptomycin by bleaching and retarded growth but no cell death. A new genetic marker for plant cell transformation has been developed that is based on the expression of the enzyme streptomycin phosphotransferase (SPT), and confers the ability to form green colonies on a selective medium. Coding sequences of SPT from the bacterial transposon Tn5 were placed under the control of gene expression signals derived from the Agrobacterium Ti plasmid Ach5. The 5 end of the SPT gene has been replaced with the promoter region of the gene coding for the first enzyme of agropine biosynthesis, the 3 end with that of the enzyme octopine synthase. The chimeric SPT gene has been linked to a selectable kanamycin resistance gene, and introduced into Nicotiana tabacum and Nicotiana plumbaginifolia by selection for the linked kanamycin resistance marker. Streptomycin resistance was expressed in some but not all of the kanamycin-resistant lines and was transmitted to the seed progeny as a dominant nuclear trait.     

Development of Improved Adenosine Deaminase Retroviral Vectors

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA⁻) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA⁻ severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.Retroviral vectors have been the most common gene transfer vehicles in clinical gene therapy trials (15). These vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells (20). The first generation of retroviral vectors have been useful in demonstrating the feasibility of gene therapy approaches, but vectors capable of higher levels of gene transfer and transgene expression would be beneficial. For example, gene transfer levels achieved by first-generation retroviral vectors in large mammals (28) and in human gene therapy trials (7, 13) have been disappointing. There are at least two avenues for improving retroviral vectors. First, molecular changes can be made in the retroviral vector sequence. Second, different packaging cell lines could be tested to modify the host range, increase transduction in a given cell type, and/or render the virions resistant to inactivation by human complement.A clinically useful model for improving retroviral vector design is the vector LASN packaged in the amphotropic line PA317. PA317/LASN was the first therapeutic vector used in a gene therapy clinical trial (1). This vector has yielded gene transfer levels of generally less than 10% in peripheral blood T cells of adenosine deaminase-deficient (ADA⁻) severe combined immunodeficiency (SCID) patients. Two possibilities to improve this vector include eliminating the dominant selectable marker gene and changing the long terminal repeat (LTR) promoter to optimize expression. LASN, like many of the retroviral vectors used in clinical trials to date, contains two genes: the therapeutic gene (the ADA gene) and a dominant selectable marker gene (the bacterial neomycin phosphotransferase II gene; neo). Dominant selectable marker genes have historically been included to facilitate the generation, isolation, and titration of retroviral producer cell clones and to permit the evaluation and selection of successfully targeted cells. neo is the most commonly used selectable marker gene, although other genes have been used, including a mutant dihydrofolate reductase gene (dhfr) (19), the multidrug resistance gene (mdr) (10), and genes for cell surface markers such as cd24 (24) and the human nerve growth factor receptor (2). Vectors carrying dominant selectable marker genes, particularly those of nonhuman origin, have two theoretical disadvantages. First, careful analysis of some patients has revealed an immune response directed against the dominant selectable marker protein expressed from the retroviral integrant (20a, 25). Second, the more complex retroviral genomes required to express two separate genes may result in lower titers or suboptimal expression of the therapeutic gene product due to promoter interference (8, 29). On the other hand, cloning and determining the titers of useful retroviral vectors without selectable markers have been laborious. Using a recently developed rapid-screening procedure, we have been able to identify a number of “simple” ADA retroviral vectors which lack dominant selectable markers (23).Different packaging cell lines may also improve gene transfer of retroviral vectors into specific target cells. Retroviral vectors are limited by the host range specified by the envelope protein on the surface of the retrovirus. Most gene therapy trials have used retroviruses with a murine amphotropic (4070A) host range. However, packaging cell lines with the gibbon ape leukemia virus (GALV) envelope (PG13 cells) (18) and the cat endogenous virus RD114 envelope (FLYRD18 cells) (5) have become available; these may improve transduction frequencies into various target cell populations. For example, there is evidence that GALV-pseudotyped retroviral vectors may facilitate gene transfer into human peripheral blood T cells with greater efficiency than vectors with an amphotropic envelope (3). Packaging cell lines derived from murine cells have the additional disadvantage that they produce retroviruses which are inactivated by complement in human sera. Packaging cell lines of human origin (FLYA13 and FLYRD18) (5) produce vectors which are complement resistant. Testing both new simple retroviral vector designs and new packaging cells may therefore improve retrovirus-mediated gene transfer.We report the construction and characterization of three simplified ADA vectors by using either the Moloney murine leukemia virus (MLV) LTR, the myeloproliferative sarcoma virus (MPSV) LTR, or the SL3-3 LTR. We tested these vectors to determine which LTR provided the highest level of ADA expression in our target cells of interest: human ADA⁻ lymphohematopoietic cells. The ADA retroviral vector with the highest level of transduction/expression was then evaluated in different packaging cell lines including PG13, FLYA13, and FLYRD18.