Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status

There are now many mammalian examples in which single cell assays of transgene activity have revealed variegated patterns of expression. We have previously reported that transgenes in which globin regulatory elements drive the lacZ reporter gene exhibit variegated expression patterns in mouse erythrocytes, with transgene activity detectable in only a sub-population of circulating erythroid cells. In order to elucidate the molecular mechanism responsible for variegated expression in this system, we have compared the chromatin structure and methylation status of the transgene locus in expressing and non-expressing populations of erythrocytes. We find that there is a difference in the chromatin conformation of the transgene locus between the two states. Relative to active transgenes, transgene loci which have been silenced exhibit a reduced sensitivity to general digestion by DNase I, as well as a failure to establish a transgene-specific DNase I hypersensitive site, suggesting that silenced transgenes are situated within less accessible chromatin structures. Surprisingly, the restrictive chromatin structure observed at silenced transgene loci did not correlate with increased methylation, with transgenes from both active and inactive loci appearing largely unmethylated following analysis with methylation-sensitive restriction enzymes and by sequencing PCR products derived from bisulphite-converted genomic DNA.     

LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.     

Gene silencing results in instability of antibody production in transgenic plants

The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed.     

Resolution of derivatives of 1,2-propanediol with lipase B from Candida antarctica. Effect of substrate structure, medium, water activity and acyl donor on enantiomeric ratio

In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20. The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E. coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER). Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells. Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene. Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells. Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal. Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines. We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors. The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours.     

The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system

Two transgenic mouse models were used to examine the cellular localization of angiotensinogen (AGT) in the brain. The first model was previously described in detail and consists of a human AGT genomic transgene containing all exons and introns of the gene and 1. 2 kb of the 5' flanking DNA. The second model contains a fusion between 1.2 kb of HAGT 5' flanking DNA and the beta-gal reporter gene which exhibits a similar pattern of tissue-specific expression to the HAGT transgene. Expression of both transgenes qualitatively mirrors the expression of endogenous AGT. Double staining of transgenic mouse brain sections with X-gal and GFAP revealed that a majority of beta-gal activity was localized to astrocytes in almost all brain areas. However, both beta-gal activity as identified by X-gal, and HAGT mRNA as detected by in situ hybridization, were also found in neurons in restricted areas of the brain, including the mesencephalic trigeminal nucleus (meV), subfornical organ (SFO) and the external lateral parabrachial nucleus (elPB). The expression of these transgenes provides the first convincing evidence for AGT gene expression in neurons in the brain. We further report by angiotensin II (Ang-II) immunostaining in rat brains after selective lesioning, that Ang-II is likely involved in a neuronal pathway from the PB to the amygdala (Ce). Finally, we performed double-labeling, first by retrograde labeling of HRP injected into the Ce, and then by X-gal on PB neurons in beta-gal transgenic mice, and identified doubly labeled neurons. Based on these results, we propose that AGT is generated in neurons in the elPB, transported to the Ce and converted into Ang-II locally to exert is biological functions.     

A composite CMV-IE enhancer/beta-actin promoter is ubiquitously expressed in mouse cutaneous epithelium

In most existing transgenic mouse models developed for the study of specific genes in the skin, the goal has been to target transgene expression to defined populations of cells in the cutaneous epithelium. Keratin promoters have been especially useful for this purpose. In some instances, however, it may be desirable to express a transgene in all the cells of the cutaneous epithelium. Since no ubiquitously expressed promoter sequences had previously been identified, we used lacZ reporter transgenes to test two enhancer/promoter sequences for ubiquitous expression in the skin of adult transgenic mice. We find that a CMV enhancer/CMV promoter is not active in most cell types in the skin, whereas a CMV enhancer/modified beta-actin promoter sequence is active in the suprabasal and basal cells of the epidermis as well as in the epithelial cells of the hair follicles, sebaceous glands, and the dermal papillae.     

Dicistronic LacZ and alkaline phosphatase reporter constructs permit simultaneous histological analysis of expression from multiple transgenes

We report here the development of convenient dicistronic transgenic markers for the rapid and efficient simultaneous analysis of transgene activity in transgenic mice. Two sensitive histological markers, the beta-galactosidase (beta-gal)-encoding lacZ gene and the human placental alkaline phosphatase (hpAP) gene, have been fused to the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus, which directs efficient mRNA cap-independent entry of the translation apparatus in mammalian cells. The IRES permits efficient translation of either lacZ or hpAP when placed anywhere within transgene exonic sequences, including both 5' and 3' untranslated regions. In addition, the production of constructs for transgenic analysis of DNA regulatory elements is greatly facilitated with IRES-lacZ or IRES-hpAP, since the IRES relieves the need for complicated in frame transgene protein fusions to produce a functional beta-gal or hpAP protein.     

Sheltering of gamma-globin expression from position effects requires both an upstream locus control region and a regulatory element 3' to the A gamma-globin gene

Integration position-independent expression of human globin transgenes in transgenic mice requires the presence of regulatory elements from the beta-globin locus control region (LCR) in the transgene construct. However, several recent studies have suggested that, while clearly necessary, such elements are not by themselves sufficient to realize this effect. In the case of the human fetal gamma-globin genes, previous results have indicated that additional regulatory information required for sheltering of gamma-globin transgene expression from position effects may reside downstream from the A gamma gene. To investigate this possibility, we established 17 lines of transgenic mice carrying constructs comprising a micro-LCR (microLCR) element, an A gamma-globin gene fragment, and a variable length of 3' sequence information beyond the A gamma 3' HindIII site. gamma-Globin expression during development was studied in 170 individual F2 progeny from these lines. We find that gamma-globin expression becomes sheltered from position effects when the normally position-sensitive microLCR-A gamma construct is extended by 600 bp beyond the 3' HindIII site to include a previously identified regulatory sequence (the A gamma-globin enhancer), the functional significance of which in vivo had heretofore been unclear. The results suggest that the mechanism whereby an upstream LCR achieves sheltering of globin gene expression from position effects involves cooperation with a gene-proximal regulatory element distinct from the promoter region.     

The Hytrin Community Assessment Trial study: a one-year study of terazosin versus placebo in the treatment of men with symptomatic benign prostatic hyperplasia. HYCAT Investigator Group

Functional differentiation of mammary tissue progresses in distinct phases spanning puberty and pregnancy. Here we have analyzed and compared the effects of transforming growth factor beta 1 (TGF beta 1), TGF alpha, and whey acidic protein (WAP), the Notch-related cell fate protein Int3, and p53 and pRb on mammary development. We chose transgene expression from the WAP gene promoter which is only active in mammary alveolar cells. The imbalanced expression of these molecules specifically altered development and differentiation of the gland. While TGF alpha did not disturb alveolar outgrowth, little or no alveolar structures developed in the presence of Int3. TGF beta 1, WAP, and the expression of SV40 T-antigen-which inactivates p53 and PRb-reduced overall alveolar development. The expression of individual milk protein genes was affected differentially by the transgenes. A WAP-lacZ transgene served as an additional indicator of terminal differentiation of alveolar cells, Homogeneous expression of lacZ was seen in mice transgenic for lacZ, or for TGF alpha and lacZ. In contrast, only a few differentiated cells were observed in the presence of TGF beta 1 and Tag. Thus, the expression of growth regulators in the same defined subset of mammary cells results in distinct developmental changes and a specific pattern of alveolar differentiation.     

Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes

We generated plasmid expression vectors encoding ubiquitin and beta-galactosidase (beta-gal) with different intervening amino acids, allowing for the production of processed protein products that have either stabilizing or destabilizing residues at their N-termini. P815 cells transfected with plasmids encoding beta-gal with a destabilizing N-terminus did not have detectable expression beta-gal unless they were treated with inhibitors specific for the proteasome. Inhibitors of other proteolysis pathways had no such effect. Nevertheless, transfectants expressing beta-gal with different amino acid residues were equally sensitive to cytolysis by a CTL clone specific for a beta-gal peptide presented in the context of H-2Ld. In contrast to vectors encoding native beta-gal, plasmid vectors encoding beta-gal with a destabilizing residue did not induce detectable anti-beta-gal Abs when injected into skeletal muscle of BALB/c mice. However, such vectors were significantly more effective than vectors encoding native beta-gal or beta-gal with a stabilizing residue in stimulating CTL specific for P13.2, a lacZ transfectant of P815. We conclude that incorporation of strategies that enhance proteasome-dependent degradation may generate DNA vaccines that are more effective in inducing cellular immunity against targeted Ags.     

Trichoverroid stereoisomers

An adeno-associated virus vector containing a lacZ gene driven by a CMV immediate-early promoter (AAV beta-gal) was evaluated with respect to its transduction efficiency and integration ability in nondividing human NT neurons. A dose-dependent pattern in transduction efficiency of the AAV beta-gal was demonstrated immunocytochemically, with up to 100% of the neurons expressing the gene product. No neurotoxic effects of the vector were detected. Quantitative PCR analyses of high molecular weight cellular DNA from the transduced neurons indicated that the copy number of the AAV beta-gal genome increased gradually in a time dependent manner, suggesting a slow but progressive rate of vector integration over a period of approximately 1 week following transduction. Equal or greater transduction efficiency of the AAV beta-gal into NT neurons than into a standard target cell line indicated that the NT neurons were readily susceptible to AAV-mediated gene transfer. This study demonstrates that AAV-based vectors can efficiently transduce and stably express a foreign gene in post-mitotic human neurons.     

Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.     

Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovirus vectors delivered to mouse liver

The activation of cytotoxic T lymphocytes (CTLs) to cells infected with adenovirus vectors contributes to problems of inflammation and transient gene expression that attend their use in gene therapy. The goal of this study was to identify in a murine model of liver gene therapy the proteins that provide targets to CTLs and to characterize the major histocompatibility complex (MHC) class I restricting elements. Mice of different MHC haplotypes were infected with an E1-deleted adenovirus expressing human alkaline phosphatase (ALP) or beta-galactosidase as a reporter protein, and splenocytes were harvested for in vitro CTL assays to aid in the characterization of CTL epitopes. A library of vaccinia viruses was created to express individual viral open reading frames, as well as the ALP and lacZ transgenes. The MHC haplotype had a dramatic impact on the distribution of CTL targets: in C57BL/6 mice, the hexon protein presented by both H-2Kb and H2Db was dominant, and in C3H mice, H-2Dk-restricted presentation of ALP was dominant. Adoptive transfer of CTLs specific for various adenovirus proteins or transgene products into either Rag-I or C3H-scid mice infected previously with an E1-deleted adenovirus verified the in vivo relevance of the adenovirus-specific CTL targets identified in vitro. The results of these experiments illustrate the impact of lr gene control on the response to gene therapy with adenovirus vectors and suggest that the efficacy of therapy with adenovirus vectors may exhibit considerable heterogeneity when applied in human populations.     

Regulation of insulin-like growth factor I (IGF-I) gene expression in brain of transgenic mice expressing an IGF-I-luciferase fusion gene

Insulin-like growth factor I (IGF-I) plays an important role in the development and function of the central nervous system (CNS). Little is known, however, about the factors and mechanisms involved in regulation of CNS IGF-I gene expression. To facilitate our goal to define mechanisms of IGF-I gene regulation in the CNS, we generated several lines of transgenic (Tg) mice that express firefly luciferase (LUC) under control of a 11.3-kb fragment from the 5' region of the rat IGF-I gene. Consistent with expression of the native IGF-I gene in murine brain, expression of the transgene predominated in neurons and astrocytes and used promoter 1, the major IGF-I promoter in the CNS and in most tissues. Transgene messenger RNA and protein expression rapidly increased after birth and peaked at postnatal (P) day 4 in all brain regions studied. LUC activities in all regions then gradually decreased to 0.5-4% of their peak values at P31, except for the olfactory bulb, which maintained about one third of its maximal activity. Compared with littermate controls, administration of dexamethasone decreased LUC activity and transgenic IGF-I messenger RNA abundance, whereas GH significantly increased the expression of the transgene. Addition of GH to cultured fetal brain cells from Tg mice for 12 h also increased LUC activity in a dose-dependent manner (77-388%). These results show that this IGF-I promoter transgene is expressed in a fashion similar to the endogenous IGF-I gene, and thus indicates that the transgene contains cis-elements essential for developmental, GH, and glucocorticoid regulation of IGF-I gene expression in the CNS. These Tg mice should serve as an useful model to study mechanisms of IGF-I gene regulation in the brain.     

prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme

The paired-related homeobox gene, prx-1, is expressed in the postmigratory cranial mesenchyme of all facial prominences and is required for the formation of proximal first arch derivatives. We introduced lacZ into the prx-1 locus to study the developmental fate of cells destined to express prx-1 in the prx-1 mutant background. lacZ was normally expressed in prx-1(neo); prx-1(lacZ )mutant craniofacial mesenchyme up until 11.5 d.p.c. At later time points, lacZ expression was lost from structures that are defective in the prx-1(neo) mutant mice. A related gene, prx-2, demonstrated overlapping expression with prx-1. To test the idea that prx-1 and prx-2 perform redundant functions, we generated prx-1(neo;)prx-2 compound mutant mice. Double mutant mice had novel phenotypes in which the rostral aspect of the mandible was defective, the mandibular incisor arrested as a single, bud-stage tooth germ and Meckel's cartilage was absent. Expression of two markers for tooth development, pax9 and patched, were downregulated. Using a transgene that marks a subset of prx-1-expressing cells in the craniofacial mesenchyme, we showed that cells within the hyoid arch take on the properties of the first branchial arch. These data suggest that prx-1 and prx-2 coordinately regulate gene expression in cells that contribute to the distal aspects of the mandibular arch mesenchyme and that prx-1 and prx-2 play a role in the maintenance of cell fate within the craniofacial mesenchyme.     

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.