Showing papers in "Transgenic Research in 1993"

New Agrobacterium helper plasmids for gene transfer to plants

Abstract: We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these ‘disamed’ Ti plasmids for plant transformation viaAgrobacterium are discussed.

Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis

Abstract: The yield and assembly of an IgG1 antibody and its derived Fab fragment were compared inNicotiana andArabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and Fab accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the Fab fragment in leaf tissue of regenerated plants differed significantly betweenNicotiana andArabidopsis. The Fab fragment accumulated to only 0.044% of TSP inNicotiana leaves but up to 1.3% inArabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. WhereasArabidopsis contained primarily fully assembled antibodies of 150 kDa,Nicotiana showed an abundance of fragments in the 50 kDa range.

Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants

Abstract: As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterlessuidA (gus A) gene, encoding the β-glucuronidase (GUS) reporter, was introduced into populations of tobacco,Arbidopsis and potato byAgrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of thegusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns ofgusA activation in diverse organs and cell types in those populations.gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency ofgusA activation was differentially blased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T2 and T3 progeny. Developmentally-regulated and environmentally-inducible expression ofgusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.

Frequency and distance of pollen dispersal from transgenic oilseed rape ( Brassica napus )

Abstract: The objective of this study was to evaluate pollen dispersal inBrassica napus (oilseed rape). The selectable marker, used to follow pollen movement, was a dominant transgene (bar) conferring resistance to the herbicide glufosinate-ammonium. Transgenic and non-transgenic plants of the cultivar Westar were planted in a 1.1 ha field trial, with the transgenic plants in a 9 m diameter circle at the centre, surrounded by non-transgenic plants to a distance of at least 47 m in all directions. A 1 m circle of non-transgenic plants was sown in the centre of the transgenic area to allow estimation of the level of pollen dispersal when plants were in close contact. Honeybee hives were placed at the trial site to optimize the opportunity for cross-pollination. During the flowering period, regular observations were made of the number of plants flowering and the number and type of insects present in 60 1 m2 areas. These areas were located uniformly around the plot at distances of 1, 3, 6, 12, 24, 36 and 47 m from the edge of the 9 m circle of transgenic plants. Seed samples were harvested from each of the 7 distances so that approximately 20% of the circumference of the plot was sampled at each distance. The centre non-transgenic circle was also sampled. Plants were grown from the seed samples and sprayed with glufosinate to estimate the frequency of pollen dispersal at each distance. In order to screen enough samples to detect low frequency cross-pollination events, seed samples were tested in the greenhouse and on a larger scale in the field. Results were confirmed by testing progeny for glufosinate resistance and by Southern blot analysis. The estimated percentage of pollen dispersal in the non-transgenic centre circle was 4.8%. The frequency was estimated to be 1.5% at a distance of 1 m and 0.4% at 3 m. The frequency decreased sharply to 0.02% at 12 m and was only 0.00033% at 47 m. No obvious directional effects were detected that could be ascribed to wind or insect activity.

Glucoamylase overexpression in Aspergillus niger: molecular genetic analysis of strains containing multiple copies of the glaA gene.

Abstract: A strategy, based on the usage of the amdS selection marker and a cosmid vector containing four copies of the glucoamylase gene (glaA), was developed to obtain glucoamylase (GLA)-overproducing A. niger strains. With this strategy, fungal strains carrying up to 200 copies of the glaA gene could be isolated at a relatively high frequency. In each transformant analysed, integration occurred in a single chromosome. A significant increase in the extracellular GLA production was observed in most of the transformants carrying multiple copies of the glaA gene. Further analysis showed that the amount of GLA that is produced was not proportional to the number of glaA copies in these transformants. However, the level of GLA production clearly correlated with the amount of glaA mRNA produced in these transformants. From these results it is concluded that GLA production is limited at the level of transcription.

An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

Abstract: A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SARL fragment either at one side (5′ or 3′) or at both sides of a heat shock promoter-regulated β-glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible β-glucuronidase activity was observed in transgenic tobacco plants containing constructs with SARL fragments either at both sides or with at least one SARL copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.

The majority of G0 transgenic mice are derived from mosaic embryos

Abstract: Most transgenic mice are generated by the direct microinjection of DNA fragments into the pronuclei of fertilized eggs. It has been generally assumed that the majority of integration events occur prior to the first round of chromosomal DNA replication (Palmiter and Brinster, 1986). In this study we have determined by comparison of PCR, Southern blot and transmission frequencies that at least 62% of integration events generate a mosaic (somatic and/or germline) G0 transgenic mouse. Furthermore, the statistical probability of transgene-containing cells segregating to the various early embryo lineages implies that this is probably an underestimate of the true mosaic frequency. Thus, the majority of DNA injected into fertilized mouse eggs intergates after the first round of chromosomal DNA replication, therefore most G0 transgenic mice are derived from a mosaic embryo.

Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells

Abstract: The diphtheria toxin A-chain gene was used in a positive-negative selection gene targeting vector to alter the CD4 gene which is transcriptionally silent in mouse embryonic stem cells. Expression of the toxin gene was driven by a constitutively active enhancer, yet the targeting construct exhibited only minimal transient toxicity while enriching for targeted clones 9- to 29-fold. Germline transmissiion of the stem cell-derived genome was obtained. These data suggest the usefulness of this diphtheria toxin A-chain cassette in replacement-type positive-negative selection vectors. Its potential for novel applications, particularly in the enrichment for ‘hit-and-run’ insertion-type vectors, is discussed.

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

Abstract: Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of β-carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.

Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4-dichlorophenoxyacetic acid

Abstract: The agronomic performance of broad leaved crop plants such as cotton would be greatly improved if genetically-engineered resistance to broadleaf herbicides could both protect the plants from accidental spray drift damage and allow the suppression of problem broadleaf weeds by chemical means. Followingin vitro modification and the addition of plant expression signals, the gene for 2,4-D monooxygenase, a bacterial enzyme that degrades the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), was introduced into cotton plants byAgrobacterium-mediated transformation. First generation homozygous progeny of regenerated transgenic cotton plants carrying this gene exhibited up to a 50–100 fold increase in tolerance to 2,4-D compared with untransformed controls, and glasshouse trials suggest that the genetically-engineered plants would be completely protected from spray drift of 2,4-D, at least up to the normal field application rates commonly used on neighbouring cereal crops.

Agrobacterium tumefaciens-mediated expression of gusA in maize tissues

Abstract: To develop a system forAgrobacterium-mediated transformation of maize (Zea mays L.), we have investigated histochemically the transient expression of β-glucuronidase (GUS) activity in maize seedling tissue segments using binary vectors that allow minimal (pKIWI105 and pCNL1) or undetectable (p35S-GUS-INT and pCNL56) levels of GUS activity inA. tumefaciens. Tissue segments from three- to five-day-old sterile seedlings of maize genotype A188 were inoculated withA. tumefaciens. Four days after inoculation, transient expression of GUS activity was found in mesocotyl segments originating from the intercalary meristem region. This GUS activity was specific to the vascular cylinder and was not found in the internal cortical or epidermal layers, nor was it found in mature mesocotyl tissue (segments 5 mm below the coleoptilar node). Transient GUS activity was also detected in leaf and coleoptile tissues of shoot segments, but not in the shoot apexper se or in leaves younger than the first leaf. Maize tissues inoculated withA. tumefaciens strains that harbourgusA-containing binary vectors but no Ti-plasmid did not show GUS activity, supporting evidence from previous work thatvir gene activity was essential for the observed GUS activity.A. tumefaciens strains containing different types of Ti-plasmids were also tested. A strain harbouring an agropine-type Ti-plasmid was the most effective for expressing GUS activity in mesocotyl segments, whereas a strain harboring a nopaline-type Ti-plasmid was most effective for expression of GUS activity in the apical meristem-containing segment. These results indicate that different interactions occurred between the differentA. tumefaciens strains and the susceptible plant tissues. Maize genotype specificity for GUS activity in mesocotyl tissues was observed; variations in the cocultivation medium had a profound effect on the frequency of expression of GUS activity.

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

Abstract: The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial β-glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T0 generation) and their progenies (T1, T2 and T3) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and β-glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T3 generation).

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

Abstract: The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.

The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: a comparison with radioactivity

Abstract: A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2′-spiroadamantane)-4-methoxy-4(3″ phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.

Transgenesis in chickens.

Abstract: The application of transgenic technology to domestic poultry offers an alternative means to conventional practice for improvement of this highly productive agricultural species. The hen's reproductive system has unique characteristics which have imposed limitations on the use of established methods for artificial gene transfer. In this article, we review the various strategies that have been adopted to overcome the problem. Target sites for gene insertion include the fertilized ovum, the blastodermal embryo in the unincubated egg, and the primordial germ cells. Notable success in obtaining somatic and germline transformation has been achieved with the use of retroviral vectors to infect the blastodermal embryo. Current attempts to introduce DNA directly into the genome, without resort to pathogen-derived vectors, are discussed.

Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice.

Abstract: Transgenic mice were produced, carrying hybrid genes comprised of the ovine β-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences.In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2-to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction.

Abstract: We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

Abstract: The ease of integrative transformation with foreign genes and the extent of their expression and stability in successive generations determine the applicability of direct gene transfer. InNicotiana plumbaginifolia, one to ten copies of foreign DNA were integrated into the plant genome, resulting in simple to complex patterns of integration. Genetic analysis showed that in more than 50% of the cases, this DNA inserted at two or more loci in the genome. Of the 156 crosses performed between F1 monogenic transformants, only eight combinations showed linkage of the inserted neomycin phosphotransferase genes (npt). The following instability events were registered: physical loss, alterations in the initial segregation rates in successive meiotic generations observed in either selfing or crossing (reduction or increase in number of segregating loci) and genomic disorders in crosses between transformants. Among them of particular interest were the “discordant” segregation values observed between corresponding R1 and F1 progenies in up to 9% of the evaluated transformants. In addition, 5% of the transformants showed a phenotypic loss of resistance. In the F3 generation, 5 out of 15 transformants exhibited instability, which was transmitted to the F4 generation. Further increases in instability rates were observed with higher numbers of insertion loci and in crosses between independent transgenic plants, reaching 100% when a trigenic partner was involved.N. plumbaginifolia exhibited more instability thanN. tabacum under equivalent experimental conditions. The molecular bases of such instability events are discussed in relation to DNA methylation, co-suppression and genomic imbalance.

Regulation of expression of transgenes in developing fish.

Abstract: The transcriptional regulatory elements of the β-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the β-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp β-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of β-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enchancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

A DNA insertional mutation results in microphthalmia in transgenic mice.

Abstract: Transgenic mice were produced by microinjection of a humanAγ-globin gene construct containing site 2 of the locus control region and theAγ-globin gene with its 3′ enhancer sequence. One transgenic mouse line 95′HS2γen91) displayed an altered phenotype when the insertion event of this transgenic line was homozygous. These animals lack the normal pigmentation seen in their hemizygous and non-transgenic littermates, thus appearing white with unpigmented eyes. In addition, their eyes are underdeveloped, consistent with the phenotype associated with mutations at themicrophthalmia (mi) locus. Backcrosses of transgenic mice withmi mutant mice result in phenotypes showing a lack of complementation, demonstrating that the site of transgene insertion is allelic withmi. Electron microscopic analysis of hair follicles and culturing of melanocytes from the skin of transgenic animals reveals an absence of cutaneous melanocytes in homozygotes and aberrant growth and morphology of the melanocytes isolated from hemizygous animals. The results presented here summarize the effects of this new allele of themi locus.

Potential use of theaux2 gene fromAgrobacterium rhizogenes as a conditional negative marker in transgenic cabbage

Abstract: An originalAgrobacterium tumefaciens-mediated transformation procedure, based on the actions of both wild type and disarmed bacterial strains, was developed. Theaux2 gene ofA. rhizogenes was introduced into a rapid-cycling genotype of cabbage (Brassica oleracea L.). Theaux2 gene product converts naphthalene acetamide into the auxin naphthalene acetic acid. Expression of this gene in the transgenic progeny grownin vitro led to an altered root phenotype. On a medium supplemented with napthalene acetamide (NAM), two of the three analysed progenies were characterized by the formation of callus instead of roots, whereas on a NAM-free medium all the plantlets from these progenies presented a normal phenotype. Expression of theaux2 gene was also assessed under horticultural conditions by sowing seeds in sand and watering them with a nutritive solution supplemented with NAM. Under these conditions, NAM inhibited the formation of a root system in transgenic plantlets and induced the death of the transgenic plantlets three to four weeks after germination. Thus,aux2 acts as a lethal conditional marker which could be used in negative selection of cabbage. Potential utilization of theaux2 gene to screen spontaneous androgenetic plants in order to transfer cytoplasmic male sterility in a single generation is discussed.

Cis effect of lacZ sequences in transgenic mice

Abstract: Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving the Escherichia coli beta-galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression in HMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of double HMG-lacZ and HMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that the lacZ sequence might interfere negatively with the expression of the adjacent HMG-cat transgene.

Degeneration of skeletal and cardiac muscles in c- myb transgenic mice

Abstract: In order to reveal cellular processes sensitive to abnormal c-myb expressionin vivo, transgenic mice were produced by introducing the c-myb nuclear proto-oncogene under the ubiquitous transcriptional regulatory unit of the cytoplasmic β-actin gene. Expression of c-myb in thymus did not cause apparent abnormality, but the mice unexpectedly developed degenerative abnormalities in skeletal and cardiac muscles; this occurred predominantly in males. Expression of c-myb in skeletal muscle was correlated with an inflammation of muscle and was accompanied by vacuolar degeneration of muscle fibres, their regeneration, and lymphocyte infiltration. The identical pathological progression in cardiac muscle was associated with cardiomegaly.

Disruption of the Gi2α locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair

Abstract: We have used an insertion vector-based approach to target the G(i2) alpha gene in AB-1 embryonic stem cells. 105 bp located 0.8-0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineered Not I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained ('hit' step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy,2-fluoro-beta-D-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination ('run' step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified 'hit and run' strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated G(i2) alpha allele is also demonstrated.

Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives.

Abstract: Transgenes in mice often exhibit different expression patterns in different transgenic lines. While the basis for this phenomenon is not understood, it is widely believed that the site at which the transgene becomes integrated into the mouse genome is a major factor in determining the pattern of expression. Most transgenic mice have been produced by microinjection of DNA into the male pronucleus, which results in integration of tandem arrays of the transgene at random chromosomal sites. In the experiments described in this report, electroporation of embryonic stem (ES) cells was used to place single copies of alacZ transgene into either random sites or into the HPRT (hypoxanthine phosphoribosyl transferase) locus of the mouse genome. Expression oflacZ was assayed by histochemical staining forEscherichia coli β-galactosidase activity in ES cells and in differentiated derivatives obtained by teratocarcinoma formation. Several of the randomly integrated cell lines expressedlacZ at high levels in a variety of cell types present in the tumours, but most notably in epithelial cells. Targeted cell lines withlacZ in opposite orientation to the direction of HPRT gene transcription also expressed well in epithelial cells, but the targeted cell lines did not express in a wider variety of cell types than some of the nontargeted cell lines. Targeted cell lines transcribinglacZ in the same orientation as HPRT transcription did not express high levels oflacZ in any differentiated cell type. Analysis of transcripts suggested that this orientation effect may have been the result of transcriptional interference perpetrated by the HPRT gene promoter.

Tobacco plants transformed with the potato virus yN coat protein gene are protected against different PVY isolates and against aphid-mediated infection

Abstract: Tobacco plant lines transformed with the coat protein (CP) gene of the tobacco veinal necrosis strain of potato virus Y (PVYN), and previously shown to be protected against mechanical inoculation with the virus, have now been tested for specificity and protection against virus infection mediated by viruliferous aphids. To determine the specificity of virus protection, two transgenic tobacco lines, A30 and A80, were challenged with several isolates of distinct PVY strains (PVYN, PVYO and PVYC) by mechanical inoculation. Clear levels of protection against the PVYO-isolates tested were maintained in the transgenic plants, although these levels were slightly lower than the protection against the homologous PVYN strain from which the CP gene was derived. Interestingly, no protection against mechanical virus inoculation with the ‘Gladblaadje’ isolate of PVYC could be observed. To assess the levels of protection against aphid-mediated virus infection, two transgenic plant lines, A30 and D25, showing respective levels of protection of 95 and 80% against mechanical virus inoculation, were challenged using PVYN viruliferousMyzus persicae. Virus inoculation using six aphids per plant, resulted in similar levels of protection in both transgenic lines as found previously for mechanical inoculation. Protection was maintained in both lines, even when as many as 60 viruliferous aphids were used per plant in the inoculation experiments.

Insect-resistant chrysanthemum calluses by introduction of a Bacillus thuringiensis crystal protein gene.

Abstract: A 3′-end truncated crystal protein gene, derived fromBacillus thuringiensis (Bt) subsp.aizawai 7.21, encoding the toxic fragment of the insecticidal proteincryIA(b), was constructed. The gene was inserted into a transformation vector, also carrying the neomycin phosphotransferase II (nptII) gene and the β-glucuronidase (gus) gene, and introduced in the oncogenicAgrobacterium tumerfaciens strain A281, harbouring the Ti-plasmid pTiBO542. The recombinantAgrobacterium strain was used to transform leaf explants of chrysanthemum (Dendranthema grandiflora) cultivar Parliament. The resulting tumours were kanamycin-resistant, exhibited β-glucuronidase activity and produced agropine and mannopine. In most tumours, all simultaneously transferred genes were expressed, owing to selection for the presence of both T-DNAs, but no correlation was found between the level of expression of the various genes. A bioassay was developed, in which larvae were fed with tumorous chrysanthemum tissue, in order to detect the effect of the transferred toxin gene on larval development. Using this bioassay with second instar larvae ofHeliothis virescens (tobacco budworm), 17 tumour lines were tested. Several of these lines proved to be strongly inhibitory to larval growth. These results indicate thatBt-based insect resistance might be used as a tool in reducing the amount of pesticides used in chrysanthemum culture.

Introduction of hygromycin resistance inLotus spp. throughAgrobacterium rhizogenes transformation

Abstract: The speciesLotus corniculatus andL. tenuis were transformed with anAgrobacterium rhizogenes binary vector, conferring resistance to the antibiotic hygromycin. Transgenic plants recovered from both species were tested for the ability of leaf-derived calluses to grow in a hygromycin-supplemented medium. Molecular analysis showed the integration of the Ri T-DNA and of the gene for hygromycin resistance, with a high frequency of co-transformation. Progeny analysis of the hygromycin resistance indicated this to be a single Mendelian trait in test plants. The transformed plants will be utilized in somatic hybridization experiments with lucerne for producing non-bloating genotypes with condensed tannins in leaves.

Use of the maize transposonsActivator andDissociation to show that phosphinothricin and spectinomycin resistance genes act non-cell-autonomously in tobacco and tomato seedlings

Abstract: Cell-autonomous genes have been used to monitor the excision of both endogenous transposons in maize andAntirrhinum, and transposons introduced into transgenic plants. In tobacco andArabidopsis, the streptomycin phosphotransferase (SPT) gene reveals somatic excision of the maize transposonActivator (Ac) as green sectors on a white background in cotyledons of seedlings germinated in the presence of streptomycin. Cotyledons of tomato seedlings germinated on streptomycin-containing medium do not bleach, suggesting that a different assay for transposon excision in tomato is desirable. We have tested the use of the spectinomycin resistance (SPEC) gene (aadA) and a Basta resistance (BAR) gene (phosphinothricin acetyltransferase, or PAT) for monitoring somatic excision ofAc in tobacco and tomato. Both genetic and molecular studies demonstrate that genotypically variegated individuals that carry clones of cells from whichAc orDs have excised from either SPEC or BAR genes, can be phenotypically completely resistant to the corresponding antibiotic. This demonstrates that these genes act non-cell-autonomously, in contrast to the SPT gene in tobacco. Possible reasons for this difference are discussed.