Homozygous Transgenic Lines Research Articles

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 μg ml−1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.

Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus.

In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.

Focal transgene expression associated with papilloma development in v‐Ha‐ras–transgenic TG.AC mice

The homozygous transgenic mouse line TG.AC contains a v-Ha-ras transgene and rapidly develops epidermal papillomas in response to either wounding or treatment with tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). The transgenic v-Ha-ras protein product was detected in all papillomas removed from TPA-treated TG.AC mice but not in vehicle- or TPA-treated TG.AC skin without tumors. In situ hybridization demonstrated that focal expression of the transgene was limited to regions of papilloma development and further localized the expression of the transgene message to the epidermal component of the papillomas, with the strongest signal in the basal epidermoid cells. Cellular proliferation, as indicated by immunohistochemical staining for proliferating-cell nuclear antigen (PCNA), was similarly localized primarily to basal epidermoid cells and, to a lesser extent, stratum spinosum cells in all papillomas analyzed. Cells that stained positively for PCNA were much more common in the papillomas than in the surrounding, normal-appearing skin. The focal nature of papilloma development was also evidenced by protein kinase C activity and hyperplasia after TPA treatment. As early as 18 d after the start of TPA treatment, focal hyperplasias associated with the follicular epidermis were observed in TG.AC but not nontransgenic FVB/N skin; these hyperplasias were assumed to be the precursors of the epidermal papillomas. To explain the development of transgene-expressing tumors from apparently transgene-negative, normal-appearing skin, we hypothesize that the papillomas arise from the clonal expansion of focal areas of epidermal cells that overexpress the transgene. We also propose that the TG.AC line is an excellent model for studying very early events in papillomagenesis.

The human osteocalcin promoter directs bone-specific vitamin D-regulatable gene expression in transgenic mice.

Osteocalcin is a major noncollagenous protein of bone regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and is believed to be expressed only by differentiated osteoblasts. We introduced a 3.9-kilobase human osteocalcin gene promoter (hOCP)-chloramphenicol acetyltransferase (CAT) fusion gene into the germ line of mice. Examination of tissue extracts from these transgenic mice demonstrated that the expression of CAT was restricted to bone-associated tissues and the brain. Immunohistochemical staining of femur tissue sections using CAT antibodies localized the production of CAT protein to osteoblasts and maturing chondrocytes. Previous studies via transient transfection into osteoblast-like cells have identified a vitamin D response element approximately 500 basepairs up-stream of the hOCP capable of mediating 1,25-(OH)2D3 induction. As a consequence, regulation of the transgene was examined in homozygous transgenic lines for sensitivity to 1,25-(OH)2D3. Hormonal deficiency was created using a low calcium diet supplemented with 0.8% SrCl2 for 7 days and was restored in experimental mice by injection of 25 ng 1,25-(OH)2D3/day, ip, for 3 days. The low vitamin D3 diet decreased CAT activity several-fold in extracts from calvaria, femur, and brain compared to that in mice maintained on a normal diet, while 1,25-(OH)2D3 supplementation restored and enhanced CAT activity over control values. These data demonstrate that hOCP is sufficient to direct osteoblast-specific 1,25-(OH)2D3-sensitive gene expression in mice in addition to the unexpected regulatable expression in brain tissue.

Spatial and temporal expression of the I factor during oogenesis in Drosophila melanogaster.

The I factor is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster. Its mobility is associated with the I-R hybrid dysgenesis. In order to study the expression pattern of this LINE in vivo, a translational fusion between the first ORF of the I factor and the lacZ gene of Escherichia coli has been carried out and introduced in the genome of reactive (R) flies. Homozygous transgenic Drosophila lines have been established and analysed. ORF1 expression is limited to germ-line cells (nurse cells and oocyte) between stage 2 and 10 of oogenesis. No somatic expression is found. Position effects may limit the level of expression of a given transgene but do not modify its basic pattern of expression during the development of the fly. This reproducible control demonstrates both that I factor is driven by its own promoter, probably the internal one suggested by Mizrokhi et al. (Mizrokhi, L.J., Georgevia, S.G. and Ilying, Y.V. (1988). Cell 54, 685-691), and that tissue-specific regulatory sequences are present in the 5' untranslated part of the I factor. The nuclear localization of the fusion protein reveals the presence of nuclear localization signals (NLS) in the ORF1-encoded protein correlating with the possible structural and/or regulatory role of this protein. This expression is restricted to dysgenic and reactive females, and is similar in the two conditions. All the results obtained in this work suggest that I factor transposition occurs as a meiotic event, between stage 2 and 10 of the oogenesis and is regulated at the transcriptional level. It also appears that our transgene is an efficient marker to follow I factor expression.

Photoresponses of transgenic tobacco plants expressing an oat phytochrome gene

The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005-1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll a∶b ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R ∶ FR. Interpretation of these data is difficult. The depression of the chlorophyll a∶b ratio by low R ∶ FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.