Transgenic markers for mammalian chimeras

Summary We describe the construction of aggregation chimeras between normal and transgenic embryos containing multiple copies of mouse -globin genes. The transgenic component of the chimeras is then detected in tissue sections by a DNA-DNA in situ hybridization technique, using a biotinylated DNA -globin probe and an avidin-linked alkaline phosphatase detection system. The general advantages of transgenic markers for chimeras are discussed.     

Efficiency of semi-automated fluorescent multiplex PCRs with 11 microsatellite markers for genetic studies of deer populations

Thirty bovine and eight ovine microsatellite primer pairs were tested on four tropical deer species: Eld's and Swamp deer (highly threatened) and Rusa and Vietnamese Sika deer (economically important). Thirty markers gave an amplified product in all four species (78.9%). The number of polymorphic microsatellite markers varied among the species from 14 in Eld's deer (47%) to 20 in Swamp deer (67%). Among them, 11 microsatellite loci were multiplexed in three polymerase chain reactions (PCRs) and labelled with three different fluorochromes that can be loaded in one gel-lane. To test the efficiency of the multiplex, primary genetic studies (mean number of alleles, expected heterozygosities and Fis values) were carried out on four deer populations. Parentage exclusion probability and probability of identity were computed and discussed on a Swamp deer population. These multiplexes PCRs were also tested on several other deer species and subspecies. The aim of this study is to establish a tool useful for genetic studies of population structure and diversity in four tropical deer species which with few modifications can be applied to other species of the genus Cervus.     

Some fluorescent counterstains for neuroanatomical studies

Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ultraviolet, light; since the tracers fluoresce only when exposed to ultraviolet light, they are not masked by the counterstain. In addition, counterstaining at pH 2 increases bisbenzimide fluorescence considerably. Ethidium bromide is useful for immunohistochemistry (IHC) because it yields a bright red Nissl counterstain when excited by green light, and is only faintly visible when the fluorescein marker is excited with blue light, or when ultraviolet excitation is used. Ethidium bromide is therefore a good counterstain for fluorescent retrograde tracer and for combined IHC-retrograde tracer studies as well. Certain dyes are also useful for studies of the normal morphology of neural tissue. For example, bisbenzimide and nuclear yellow at low concentrations produce a brilliant Nissl stain at pH 2, and stain only nuclei at pH 7.2. The latter procedure may be particularly useful for cell counts. Finally, neutral red, astrazone red, and safranin-O differentially stain cells amd myelinated fibers, producing fluorescence analogs of the Klüver-Barrera stain.     

Characterization of mammalian receptors for lactoferrin.

Lactoferrin (Lf) has been suggested to have several physiological functions. Specific binding of Lf, indicating the presence of Lf receptors (LfRs), has been observed in various types of mammalian cells such as lymphocytes, hepatocytes, and enterocytes. These LfRs are considered to function as a mediator for some of the functions of Lf. We here review current knowledge of mammalian LfRs characterized in different tissues. We also briefly present evidence for the existence of an LfR provided by our cloning of a human intestinal LfR (HLfR). The entire coding region of the HLfR was cloned by polymerase chain reaction (PCR), and a recombinant HLfR (rHLfR) was expressed in a baculovirus system. The rHLfR was purified by immobilized human Lf (HLf) affinity chromatography, indicating that the rHLfR retained the capacity to bind HLf. The gene was expressed at high levels in fetal small intestine and in adult heart but at lower levels in Caco-2 cells. In summary, we demonstrate the presence of a unique receptor-mediated mechanism for Lf, functioning in the small intestine of the newborn infant and possibly in other tissues of human adults.     

Characterization of sensitivity and specificity of fluorescent markers of structural state and binding ability of proteins

Methods for determining the sensitivity of fluorescent probes and their classification with respect to sensitivity and specificity using human serum albumin as a reference protein and 8-anili-nonaphthalenesulfonate, K35 and pyron red as probes are proposed. The indicators of sensitivity and specificity of fluorescent probes as markers of the structural state of proteins are the activation of the probe fluorescence in excess of protein and the constant of its binding to the strong highly affine center of protein sorption. Fluorescent probes as markers of the binding ability are assessed by a protein-dependent increase in fluorescence under the conditions of excess probe and from the constant of its binding to weak secondary binding sites of the protein.     

Characterization of nuclear compartments identified by ectopic markers in mammalian cells with distinctly different karyotype

The functional organization of chromatin in cell nuclei is a fundamental question in modern cell biology. Individual chromosomes occupy distinct chromosome territories in interphase nuclei. Nuclear bodies localize outside the territories and colocalize with ectopically expressed proteins in a nuclear subcompartment, the interchromosomal domain compartment. In order to investigate the structure of this compartment in mammalian cells with distinctly different karyotypes, we analyzed human HeLa cells (3n+=71 chromosomes) and cells of two closely related muntjac species, the Chinese muntjac (2n=46 chromosomes) and the Indian muntjac (2n=6/7 chromosomes). The distribution of ectopically expressed intermediate filament proteins (vimentin and cytokeratins) engineered to contain a nuclear localization sequence (NLS) and a nuclear particle forming protein (murine Mx1) fused to a yellow fluorescent protein (YFP) was compared. The proteins were predominantly localized in regions with poor DAPI staining independent of the cells karyotype. In contrast to NLS-vimentin, the NLS-modified cytokeratins were also found close to the nuclear periphery. In Indian muntjac cells, NLS-vimentin colocalized with Mx1-YFP as well as the NLS-cytokeratins. Since the distribution of the ectopically expressed protein markers is similar in cells with distinctly different chromosome numbers, the property of the delineated, limited compartment might indeed depend on chromatin organization.     

Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.     

New microsatellite markers in chicken optimized for automated fluorescent genotyping

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4·0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).     

Elimination of selection markers from transgenic plants

Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields. Regimes to achieve their efficient elimination, mostly through site-specific recombination or transposition, are being developed.     

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.     

A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants

Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.     

Seed-transmissible expression of mammalian metallothionein in transgenic tobacco

A binary plasmid was constructed to contain the mouse metallothionein c-DNA, the constitutive 35S promoter from cauliflower mosaic virus, the polyadenylation signal from the pea rbcS-E9 gene and several selectable markers. The plasmid was transferred to Agrobacterium tumefaciens and the leaf disc method was used to transform tobacco. Callus and shoots were regenerated in the presence of kanamycin and transformed plants were obtained. Southern, Northern and Western blot analysis demonstrated integration and expression of the metallothionein gene in transformed callus and transgenic plants. The gene is transmitted to and expressed in seed derived progeny as a dominant Mendelian trait.     

Studies with a fluorescent vital probe for DNA in mammalian cells

Olivomycin is taken up efficiently by HeLa cells and by rat fibroblast cells at 38.5 °C, but not by BHK cells. On irradiation with light of 425 nm wavelength, the nuclei of living cells that have taken up olivomycin fluoresce. When olivomycin complexes with DNA in solution, the emission spectrum broadens and shifts, the excitation wavelength maximum shifts up 15 nm, and the fluorescence polarization increases. In HeLa and fibroblast cells, the fluorescence characteristics indicate that olivomycin is entirely complexed to DNA, and its rotational mobility indicates that it is complexed to DNA in regions where other components of the chromatin offer no steric hindrance.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Transthyretin is synthesized in the mammalian eye

Transthyretin (TTR, prealbumin) is a 55 kDa protein which plays an important role in the plasma transport of thyroxine and retinol. Although the liver and choroid plexus are the two major known sites of TTR synthesis, several lines of evidence suggest the possibility of a separate ocular source of TTR. We report the presence of TTR mRNA in rat and bovine eye and of TTR in rat eye. Preliminary immunohistochemical data indicate that the retinal pigment epithelium is a major site of TTR immunoreactivity in the rat. While the functional significance of ocular TTR synthesis is unclear, TTR may be involved in the ocular translocation and processing of retinol. The finding of TTR synthesis in the eye may explain ocular involvement in the familial amyloidotic polyneuropathies.