Showing papers on "Transgene published in 1997"
TL;DR: In this paper, the authors observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to 157 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype.
2,223 citations
TL;DR: Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk and produced viable animals by nuclear transfer.
Abstract: Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.
996 citations
TL;DR: The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system, and can also be regulated by DEX in transgenic Arabidopsis plants.
Abstract: A novel chemical induction system for transcription in plants has been developed, taking advantage of the regulatory mechanism of vertebrate steroid hormone receptors. A chimeric transcription of the DNA-binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR). The GVG gene was introduced into transgenic tobacco and Arabidopsis together with a luciferase (Luc) gene which was transcribed from a promoter containing six tandem copies of the GAL4 upstream activating sequence. Induction of luciferase activity was observed when the transgenic tobacco plants were grown on an agar medium containing dexamethasone (DEX), a strong synthetic glucocorticoid. Induction levels of the luciferase activity were well correlated with DEX concentrations in the range from 0.1 to 10 microM and the maximum expression level was over 100 times that of the basal level. Analysis of the induction kinetics by Northern blot analysis showed that the Luc mRNA was first detected 1 h after DEX treatment and increased to the maximum level in 4 h. The stationary induction level and the duration of the induction varied with the glucocorticoid derivative used. The GVG gene activity can also be regulated by DEX in transgenic Arabidopsis plants. The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system. Advantages and potential uses of this system are also discussed.
968 citations
TL;DR: In this article, it was found that nepovirus infection of nontransgenic plants induces a resistance mechanism that is similar to transgene-induced gene silencing, which may be related to natural defense against viruses.
Abstract: Gene silencing in plants, in which an endogenous gene is suppressed by introduction of a related transgene, has been used for crop improvement. Observations that viruses are potentially both initiators and targets of gene silencing suggested that this phenomenon may be related to natural defense against viruses. Supporting this idea, it was found that nepovirus infection of nontransgenic plants induces a resistance mechanism that is similar to transgene-induced gene silencing.
865 citations
TL;DR: In conclusion, the mouse overexpressing TNF-alpha recapitulated the phenotype of congestive heart failure and provides a novel model to elucidate the role of this cytokine in the development of congestives heart failure.
Abstract: The failing human heart expresses tumor necrosis factor-α (TNF-α). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-α in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-α by preserving the destabilizing sequence in TNF-α cDNA. Expression was driven by the murine α-myosin heavy chain promoter. Use of this modified construct allowed us to establish a murine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight–to–body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-α protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart...
842 citations
TL;DR: Results suggest that a non‐metabolic, transgene‐specific, diffusable messenger mediates the propagation of de novo post‐transcriptional silencing through the plant.
Abstract: Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and transgenes and of post-transcriptional silencing of a uidA transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding transgene. Transmission is unidirectional from stock to scion, transgene specific, locus independent and requires the presence of a transcriptionally active transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.
816 citations
TL;DR: SRE BP-1a is the most active form of SREBP-1 and that SREbp-1c may be produced when cells require a lower rate of transcription of genes regulating cholesterol and fatty acid metabolism.
Abstract: We have produced transgenic mice whose livers express a dominant positive NH2-terminal fragment of sterol regulatory element binding protein-1c (SREBP-1c). Unlike full-length SREBP-1c, the NH2-terminal fragment enters the nucleus without a requirement for proteolytic release from cell membranes, and hence it is immune to downregulation by sterols. We compared SREBP-1c transgenic mice with a line of transgenic mice that produces an equal amount of the NH2-terminal fragment of SREBP-1a. SREBP-1a and -1c are alternate transcripts from a single gene that differ in the first exon, which encodes part of an acidic activation domain. The 1a protein contains a long activation domain with 12 negatively charged amino acids, whereas the 1c protein contains a short activation domain with only 6 such amino acids. As previously reported, livers of the SREBP-1a transgenic mice were massively enlarged, owing to accumulation of triglycerides and cholesterol. SREBP-1c transgenic livers were only slightly enlarged with only a moderate increase in triglycerides, but not cholesterol. The mRNAs for the LDL receptor and several cholesterol biosynthetic enzymes were elevated in SREBP-la transgenic mice, but not in 1c transgenic mice. The mRNAs for fatty acid synthase and acetyl CoA carboxylase were elevated 9- and 16-fold in la animals, but only 2- and 4-fold in 1c animals. Experiments with transfected cells confirmed that SREBP-1c is a much weaker activator of transcription than SREBP-1a when both are expressed at levels approximating those found in nontransfected cells. SREBP-1c became a strong activator only when expressed at supraphysiologic levels. We conclude that SREBP-1a is the most active form of SREBP-1 and that SREBP-1c may be produced when cells require a lower rate of transcription of genes regulating cholesterol and fatty acid metabolism.
799 citations
TL;DR: The SHIRPA procedure is developed, which utilizes standardized protocols for behavioral and functional assessment that provide a sensitive measure for quantifying phenotype expression in the mouse, and can be refined to test the function of specific neural pathways, which will contribute to a greater understanding of neurological disorders.
Abstract: For an understanding of the aberrant biology seen in mouse mutations and identification of more subtle phenotype variation, there is a need for a full clinical and pathological characterization of the animals. Although there has been some use of sophisticated techniques, the majority of behavioral and functional analyses in mice have been qualitative rather than quantitative in nature. There is, however, no comprehensive routine screening and testing protocol designed to identify and characterize phenotype variation or disorders associated with the mouse genome. We have developed the SHIRPA procedure to characterize the phenotype of mice in three stages. The primary screen utilizes standard methods to provide a behavioral and functional profile by observational assessment. The secondary screen involves a comprehensive behavioral assessment battery and pathological analysis. These protocols provide the framework for a general phenotype assessment that is suitable for a wide range of applications, including the characterization of spontaneous and induced mutants, the analysis of transgenic and gene-targeted phenotypes, and the definition of variation between strains. The tertiary screening stage described is tailored to the assessment of existing or potential models of neurological disease, as well as the assessment of phenotypic variability that may be the result of unknown genetic influences. SHIRPA utilizes standardized protocols for behavioral and functional assessment that provide a sensitive measure for quantifying phenotype expression in the mouse. These paradigms can be refined to test the function of specific neural pathways, which will, in turn, contribute to a greater understanding of neurological disorders.
795 citations
TL;DR: A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos and embryogenic calli as explants, and stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis.
Abstract: A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.
743 citations
TL;DR: A critical role for GATA‐1 is revealed in megakaryocyte growth regulation and platelet biogenesis, and targeted mutation of cis‐elements can generate lineage‐specific knockout mice are illustrated.
Abstract: Transcription factor GATA-1 is essential for red blood cell maturation and, therefore, for survival of developing mouse embryos. GATA-1 is also expressed in megakaryocytes, mast cells, eosinophils, multipotential hematopoietic progenitors and Sertoli cells of the testis, where its functions have been elusive. Indeed, interpretation of gene function in conventional knockout mice is often limited by embryonic lethality or absence of mature cells of interest, creating the need for alternate methods to assess gene function in selected cell lineages. Emerging strategies for conditional gene inactivation through site-specific recombinases rely on the availability of mouse strains with high fidelity of transgene expression and efficient, tissue-restricted DNA excision. In an alternate approach, we modified sequences upstream of the GATA-1 locus in embryonic stem cells, including a DNase I-hypersensitive region. This resulted in generation of mice with selective loss of megakaryocyte GATA-1 expression, yet sufficient erythroid cell levels to avoid lethal anemia. The mutant mice have markedly reduced platelet numbers, associated with deregulated megakaryocyte proliferation and severely impaired cytoplasmic maturation. These findings reveal a critical role for GATA-1 in megakaryocyte growth regulation and platelet biogenesis, and illustrate how targeted mutation of cis-elements can generate lineage-specific knockout mice.
720 citations
TL;DR: The unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV) is described, where the rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers.
Abstract: Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.
TL;DR: It is shown that p53-dependent expression of bax is induced in slow-growing apoptotic tumours, and this is the first demonstration that Bax acts as a tumour suppressor, and the findings indicate that bax could be a component of the p 53-mediated apoptotic response in this system.
Abstract: The protein p53 is a key tumour-suppressor, as evidenced by its frequent inactivation in human cancers. Animal models have indicated that attenuation of p53-dependent cell death (apoptosis) can contribute to both the initiation and progression of cancer, but the molecular mechanisms are unknown. Although p53-mediated transcriptional activation is one possible explanation, none of the known p53-responsive genes has been shown to function in p53-dependent apoptosis. Here we test the role of the death-promoting gene bax in a transgenic mouse brain tumour, a model in which p53-mediated apoptosis attenuates tumour growth. Inactivation of p53 causes a dramatic acceleration of tumour growth owing to a reduction in apoptosis of over ninety per cent. We show that p53-dependent expression of bax is induced in slow-growing apoptotic tumours. Moreover, tumour growth is accelerated and apoptosis drops by fifty per cent in Bax-deficient mice, indicating that it is required for a full p53-mediated response. To our knowledge this is the first demonstration that Bax acts as a tumour suppressor, and our findings indicate that Bax could be a component of the p53-mediated apoptotic response in this system.
TL;DR: In vivo complementation with bacterial artificial chromosome clones expressed in transgenic mice to identify the circadian Clock gene provides a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.
TL;DR: A simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies and proper expression of the lacZ transgene in the brain has been observed.
Abstract: Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAC containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAC, and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.
TL;DR: It is shown that enforced expression of the bcl-2 gene in T-lymphoid cells (by crossing in the Emu-bcl- 2 transgene) in IL-7R alpha-deficient mice results in a significant restoration of thymic positive selection and T cell numbers and function.
TL;DR: The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.
Abstract: Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.
TL;DR: The studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells.
Abstract: Mouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations. However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology. Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells. Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alphaMyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alphaMyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein. Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells. Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.
TL;DR: Three different HLA-A2.1 monochains were engineered in which either the human or mouse β2-microglobulin (β2m) is covalently linked to the NH2 terminus of the heavy chain by a 15– amino acid long peptide, and the selected HHD construct was introduced by transgenesis in H-2Db/− β2m−/− double knockout mice.
Abstract: Three different HLA-A2.1 monochains were engineered in which either the human or mouse β2-microglobulin (β2m) is covalently linked to the NH2 terminus of the heavy chain by a 15– amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db α3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse β2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db−/− β2m−/− double knockout mice. Expression of this monochain restores a sizable peripheral CD8+ T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db−/− β2m−/− mice generate only HLA-A2.1–restricted CD8+ CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1–restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1–restricted CTL responses of vaccine interest.
TL;DR: This work describes the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene, and identifies a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors.
Abstract: TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5' flanking region of the TIE2 promoter is capable of directing beta-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.
TL;DR: A transgenic mouse line carrying the CAG-cre transgene in which the cre gene is under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter is developed, and Cre mRNA and/or protein are retained in mature oocytes irrespective of the transmission of the C AG-cre Transgene, resulting in efficient Cre-mediated recombination of paternally derived target genes upon fertilization.
TL;DR: The results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.
Abstract: To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.
TL;DR: The results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences in Caenorhabditis elegans and the additional requirement for let-858 in the larval germline.
Abstract: In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.
Journal Article•
TL;DR: The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.
Abstract: Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +1+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-+S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.
TL;DR: It is shown that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues.
Abstract: The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.
TL;DR: Subversion of normal mesenchymal-epithelial paracrine regulation through the forced misdirection of HGF/SF expression induces aberrant morphogenesis and subsequent malignant transformation of cells of diverse origin.
Abstract: Hepatocyte growth factor/scatter factor (HGF/SF) is a mesenchymally derived, multifunctional paracrine regulator possessing mitogenic, motogenic, and morphogenetic activities in cultured epithelial cells containing its tyrosine kinase receptor, Met. c-met has been implicated in oncogenesis through correlation of expression with malignant phenotype in specific cell lines and tumors. Paradoxically, however, HGF/SF can also inhibit the growth of some tumor cells. To elucidate the oncogenic role of HGF/SF in vivo, transgenic mice were created such that HGF/SF was inappropriately targeted to a variety of tissues. HGF/SF transgenic mice developed a remarkably broad array of histologically distinct tumors of both mesenchymal and epithelial origin. Many neoplasms arose from tissues exhibiting abnormal development, including the mammary gland, skeletal muscle, and melanocytes, suggesting a functional link between mechanisms regulating morphogenesis and those promoting tumorigenesis. Most neoplasms, especially melanomas, demonstrated overexpression of both the HGF/SF transgene and endogenous c-met, and had enhanced Met kinase activity, strongly suggesting that autocrine signaling broadly promotes tumorigenesis. Thus, subversion of normal mesenchymal–epithelial paracrine regulation through the forced misdirection of HGF/SF expression induces aberrant morphogenesis and subsequent malignant transformation of cells of diverse origin.
TL;DR: It is demonstrated that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene.
Journal Article•
TL;DR: Various observations associated with gene silencing are discussed and various factors seem to play a role, including DNA methylation, transgene copy number and the repetitiveness of thetransgene insert, transGene expression level, possible production of aberrant RNAs, and ectopic DNA‐DNA interactions.
TL;DR: In this article, transgenic mice expressing human α-, γ-, and β S -globin were generated and bred with knockout mice that had deletions of the murine α- and β-globin genes.
Abstract: To create mice expressing exclusively human sickle hemoglobin (HbS), transgenic mice expressing human α-, γ-, and β S -globin were generated and bred with knockout mice that had deletions of the murine α- and β-globin genes. These sickle cell mice have the major features (irreversibly sickled red cells, anemia, multiorgan pathology) found in humans with sickle cell disease and, as such, represent a useful in vivo system to accelerate the development of improved therapies for this common genetic disease.
TL;DR: Various observations associated with gene silencing are discussed and various factors seem to play a role, including DNA methylation, transgene copy number and the repetitiveness of thetransgene insert, transGene expression level, possible production of aberrant RNAs, and ectopic DNA–DNA interactions.
TL;DR: These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies and should be possible to visualize the origin and migration of any lineage-specific progenitors cells in a living embryo.
Abstract: In this study, DNA constructs containing the putative zebrafish promoter sequences of GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos. Erythroid-specific activity of the GATA-1 promoter was observed in living embryos during early development. Fluorescent circulating blood cells were detected in microinjected embryos 24 hours after fertilization and were still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continued to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish were consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies. By generating transgenic fish using constructs containing other zebrafish promoters and green fluorescent protein reporter gene, it should be possible to visualize the origin and migration of any lineage-specific progenitor cells in a living embryo.