A new method of deleting a specified sequence in transgenic lines of Drosophila melanogaster.
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Animals↗
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Transmission pattern of hobo transposable element in transgenic lines of Drosophila melanogaster.
This study is an attempt to trace the fate of hobo elements in the genomes of E strains of Drosophila melanogaster that have been transfected with pHFL1, a plasmid containing an autonomous hobo. Such long-term population studies (over 105 generations) could be very useful for better understanding the population and genomic dynamics of transposable elements and their pattern of insertions. Molecular analyses of hobo elements in the transfected lines were performed using Southern blots of XhoI-digested genomic DNAs. The complete element was observed in all six injected lines. In two lines we observed, at generation 100, two deleted elements, which did not correspond to Th1 and Th2. The results obtained by the in situ method show that the number of hybridization sites increases in each line and prove that the hobo element may be amplified in an RM genome. The hobo activity does not seem to be systematically correlated with the number of hobo elements. After generation 85, the evolution of the hobo element's insertion site number depends on the injected line. In all lines, the total number of insertions remains quite small, between 0 and 11. Hobo elements are located on each of the chromosomal arms. We describe 'hotspots'-insertion sites present in all lines and in all generations. On the 3R arm, a short inversion appeared once at generation 85.
Animals↗
Conservation and regeneration of transgenic lines of swine by semen cryopreservation and artificial insemination.
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Animals↗
Creation of transgenic lines using microparticle bombardment methods.
Introduction of exogenous DNA into Caenorhabditis elegans is important for examining the expression of altered or reporter gene constructs, rescuing mutant genes, and studying gene function in vivo. Until recently, germ-line injection was the most commonly used method for transforming C. elegans strains. This chapter describes four different microparticle bombardment methods used to transform C. elegans with exogenous DNA. We include a discussion of the advantages and disadvantages of using microparticle bombardment for transformation, list cotransformation markers that have been used successfully in microparticle bombardment experiments, and discuss transformation efficiency.
Animals↗
[Disruption of male meiosis in transgenic line tobacco res91].
The course of meiosis in male-sterile trasgenic tobacco line res91 has been analysed. Cytological analysis included visualization of the spindle and phragmoplast. Abnormal meiosis was characterized with 1) cytomixis; 2) deformation of nuclei at prophase 2 in part of the cells, and lack of spindle formation in such cells at M2; 3) desorientation of spindles in meiosis 2. This set of abnormalities allows to suppose certain disturbancies of cytoskeleton during male meiosis in res91.
Cell Nucleus↗
[Introduction of exogenous genes into animal lines: transgenic animals. 2. Genetic transformation in Drosophila].
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Animals↗
Inheritance analysis of herbicide-resistant transgenic soybean lines.
Four transgenic soybean lines generated via Agrobacterium-mediated transformation were used to analyze inheritance of the transgenes. Seed chip GUS assay and herbicide leaf painting and spraying assays were applied to test the gus reporter gene and the herbicide resistant bar selectable marker gene, respectively. Three of the four transgenic soybean lines were stably inherited in a Mendelian fashion with co-segregation of both transgenes in a 3:1 segregation ratio in the T(1) progeny, indicating that both transgenes were integrated into the same locus of the soybean genome. Homozygous transgenic progeny plants were obtained in the T(2) generation of these lines, and the transgenes were inherited in five successive generations. However, in one transgenic line, all the T(1) progeny plants showed GUS negative and herbicide sensitive. Southern blotting analysis confirmed that the transgenes were passed into the T(1) progeny, indicating that the transgenes were both silenced. To test if the transgene silencing was due to transcriptional or post-transcriptional level, Soybean mosaic virus (SMV) was inoculated on leaf tissues of the T(1) plants to test possible reverse effects on transgene silencing. Infection with SMV did not suppress transgene silencing, suggesting that transgene silencing in this transgenic line may not be due to post-transcriptional gene silencing.
Gene Silencing↗
Transgenic cell lines for detection of animal viruses.
Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses.
Animals↗
The use of transgenic cell lines for evaluating toxic metabolites of carbamazepine.
Human lymphoblastoid cell lines transgenic for human CYP450s were evaluated for the identification of toxic metabolites of the anticonvulsant drug carbamazepine (CBZ). Human CYP450 isoforms expressed by these cell lines included 1A1, 1A2, 2E1, 2A6, and 3A4. A dose-dependent inhibition of population growth from 50-200 micrograms/ml CBZ was detected by measuring cell number and respiration. The inhibition increased with the growth rate of the various lines, which correlated inversely with the presence of CYP450s, and may have been caused by CBZ itself. Cytotoxicity was observed only at the highest dose and in the line lacking transfected CYP450s. Microsomal preparations from hCYP3A4/OR cells converted CBZ into its principal oxidative metabolite, carbamazepine-10,11-epoxide (CBZ-E), at a rate of 630 pmol/min per mg protein, confirming a major role of CYP3A4 in this reaction. However, no CBZ-E (or any metabolite) was recovered from any whole-cell incubation even though hCYP3A4 cells readily converted testosterone to 6 beta-hydroxytestosterone. This suggests that differences exist between whole-cell and microsomal preparations of lymphoblastoid cells in their ability to metabolize CBZ.
Aryl Hydrocarbon Hydroxylases↗
Bone mass increase specific to the female in a line of transgenic mice overexpressing human osteoblast stimulating factor-1.
We have reported that transgenic mice overexpressing human osteoblast stimulating factor-1 (osf1) under the control of the human osteocalcin promoter have a significantly higher bone mineral content and density than nontransgenic littermates. Consequently, bone mass loss due to estrogen deficiency was compensated for in ovariectomized female mice. Here, we show that in this transgenic line, the bone mass increase was evident in female, but not male, mice, as evaluated using the ash assay, double-emission X-ray analysis, and calcein double-labeling to determine the bone formation rate. To elucidate a possible influence on gene expression, we analyzed genomic structures of the inserted transgene and its flanking regions in mouse chromosomes. The results revealed that the transgene was integrated in the mouse repetitive sequences, 234-bp-long gamma-satellite repeats, as inverted multiple (5 + 8) copies. Twelve copies at most seemed to be functional, but no direct evidence supporting female-specific mRNA synthesis of the transgene was obtained.
Animals↗
Transgene-induced RNA interference as a tool for plant functional genomics.
RNA interference (RNAi) is a powerful tool for functional genomics in a number of species. The logistics and procedures for doing high-throughput RNAi to investigate the functions of large numbers of genes in Arabidopsis thaliana and in Zea mays are described. Publicly available plasmid vectors that facilitate the stable chromosomal integration of inverted repeat transgenes that trigger RNAi have been used to generate more than 50 independent transgenic lines each in Arabidopsis and maize. Analysis of mRNA abundance of the targeted genes in independent lines transformed with distinct constructs indicates that the success of RNAi-induced silencing is gene dependent. mRNA levels were not detectably reduced for some genes, but were dramatically reduced for a number of genes targeted. A common pattern was that multiple independent lines transgenic for the same construct showed the same extent of silencing. This chapter describes the procedures used to generate and test transgenic lines mediating RNAi in Arabidopsis and maize.
Arabidopsis↗
lambda 5, but not mu, is required for B cell maturation in a unique gamma 2b transgenic mouse line.
gamma 2b transgenic mice have a severe B cell defect, apparently caused by strong feedback inhibition of endogenous H-gene rearrangement coupled with an inability of gamma 2b to provide the survival/maturation functions of mu. A unique gamma 2b transgenic line, named the C line, was found to permit B cell development. When the C line is crossed with a mu-membrane knockout line, gamma 2b+ B cells develop in the homozygous knockout. In contrast, a transgenic line representative of all the other gamma 2b lines is completely B cell deficient when mu-mem is deleted. Strikingly, the C phenotype is dominant in C x other gamma 2b transgenic line crosses. There is no evidence for higher gamma 2b transgene expression or other position effects on the transgene in the C mouse. The sequences of the three gamma 2b transgene copies in the C line are identical to that of the original transgene. These results have led to the conclusion that in the C line the transgene integration constitutively induces a gene whose expression can replace mu. To more clearly delineate the stage at which the altered phenotype of the C line is expressed, C mice were crossed onto a lambda 5 knockout background. In the absence of lambda 5, the C line produces no B cells. Since it was also found that gamma 2b can associate with the surrogate light chain (sL; lambda 5/Vpre-B), the crosses between C line gamma 2b mice and lambda 5 knockout mice suggest that gamma 2b/sL is required for B cell maturation in this mouse line. Thus, gamma 2b alone is unable to replace mu for pre-B cell survival/maturation; however, in combination with an unknown factor and the sL, gamma 2b can provide these nurturing functions.
Alleles↗
Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes.
A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T3, T4, T5) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and "added" subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots. These results indicate that the transgenic and control lines can be regarded as substantially equivalent in terms of stability of gene expression between generations and environments.
Breeding↗
Enhanced homologous recombination caused by the non-transcribed spacer of the rDNA in Arabidopsis.
The problem of the low frequency of homologous recombination observed in higher plants has been approached in several ways. Here, we report a new strategy to enhance homologous recombination in Arabidopsis. In Escherichia coli and Saccharomyces cerevisiae, hotspots that enhance homologous recombination nearby have been identified in regions close to sites associated with the blocking of DNA replication forks or with intensive transcriptional activity. In yeast, a recombination hotspot (HOT1) was found in a region spanning two non-transcribed spacers (NTS) between ribosomal RNA genes (rDNA), which contains both a replication fork barrier ( RFB) and the promoter for transcription of the 35S rRNA gene. Since rDNA has a common structure among eukaryotes, we analyzed the effect of the endogenous NTS on homologous mitotic recombination in a higher plant. We constructed transgenic lines of Arabidopsis containing this NTS and a recombination substrate, in which two 3'- and 5'-deleted uidA (beta-glucuronidase) genes with partially overlapping sequences are separated by a Hyg(r) gene. Reconstitution of functional uidA genes by homologous recombination was monitored by histochemical GUS staining. We found that recombination occurred more frequently in all organs tested in F (Fork block) lines transgenic for the NTS than in C (Control) lines without the NTS. The average number of GUS+ spots on leaves in F lines was more than nine-fold higher than in C lines. Furthermore, by genomic Southern analysis, post-recombinational molecules were detected in a transgenic line, F43, which had an extremely high number of GUS+ blue spots. These results strongly suggest that NTS-dependent enhancement of homologous recombination may be a common feature of higher plants as well as yeast.
Arabidopsis↗
Chromosome localization of the human insulin gene in transgenic mouse lines.
Three transgenic mouse lines, Tg 74, Tg 174, and Tg 171, were obtained by microinjection of an 11-kb human DNA fragment carrying the insulin gene into pronuclei of fertilized mouse eggs. The human insulin gene was expressed in all three transgenic mouse lines as shown by the presence of human C peptide in serum and urine and of human insulin transcripts in RNA prepared from pancreas. Several copies of the human DNA fragment arranged in head-to-tail arrays were present in each line. The human DNA insert was transmitted to the progeny as a single genetic locus. The chromosomal integration of the human insulin transgene was directly demonstrated by in situ hybridization to metaphase chromosomes of mitotic cells prepared from spleen and bone marrow. The insert appeared unique and located on a different chromosome in each line, namely 7 for Tg 74, 13 for Tg 174, and 18 for TG 171. Separation of DNA fragments larger than 20 kb by pulse-field electrophoresis showed that several insertion sites were present in each chromosome locus. This is the first direct evidence in transgenic mice that a gene located at various chromosome loci can be correctly expressed.
Animals↗
Transgene insertion pattern analysis using genomic walking in a transgenic mouse line.
A transgene mapping technique (Noguchi et al., Exp. Anim. 53:103-111, 2004) is described that can be used to analyze transgene integration patterns in transgenic mice. The technique was used to reveal that a transgenic mouse line (GM1-sy#116) harbored inverted and direct tandem repeats of both intact and partial pCAGGS-based transgenes in the G2 region of chromosome 1. This complicated concatenation of transgenes may have been caused by simple end-joining of DNA constructs fragmented by exposure to UV transillumination during gel-purification, and by nuclease digestion inside zygote pronuclei. The results suggest that care should be taken to avoid unwanted fragmentation during the preparation of vector constructs.
Animals↗
Transgene elimination in genetically modified dry bean and soybean lines.
Transgene elimination is a poorly studied phenomenon in plants. We made genetic and molecular studies of a transgenic dry bean line immune to bean golden mosaic geminivirus and a soybean line. In both lines, the transgenes were stable during the vegetative phase but were eliminated during meiosis. Due to its potential biotechnological value, this transgenic line was micropropagated by grafting and the vegetative copies were studied for more than two years. More than 300 plants of progeny were obtained during this period, demonstrating that the phenomenon of elimination was consistently repeated and offering an opportunity for detailed study of transgene elimination, including the characterization of the integration sites. Cloning and sequencing of the transgenic loci, reciprocal crosses to untransformed plants, genomic DNA blots, and GUS assays were performed in the transgenic lines. Based on the molecular and genetic characterization, possible mechanisms involved in transgene elimination include intrachromosomal recombination, genetic instability resulting from the tissue culture manipulations, and co-elimination of transgenes, triggered by a process of genome defense.
DNA, Plant↗
Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.
A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.
Amino Acid Sequence↗