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PubMed1996

The mechanism of somatic hypermutation studied with transgenic and transfected target genes.

Somatic hypermutation of Ig genes is beginning to be understood in molecular terms. Ig transgenes have served as model test genes and been shown to mutate, just as endogenous genes, with a peak of mutation over the VJ region and a sparing of the C region. The levels of somatic mutation appear to be related to the expression of the transgenes. DNA hypermethylation of modified Ig transgenes interferes with both expression and somatic hypermutation. Test substrates consisting of bacterial lacZ alpha or supFtRNA inserted within x transgenes were shown to be rescuable as expressible bacterial plasmids, but did not seem to be targeted. A synthetic sequence consisting of alternating restriction enzyme sites, that cannot be subject to methylation, was found to be a reliable transgenic substrate for easy assay of somatic mutation. The generation of a transgenic mouse with a x transgene in which the transcriptional promoter has been duplicated upstream of the C region has given new clues to the mutation mechanism. In this transgene, transcripts initiate from both the C region and the V region promoters, and both regions, but not the sequences between them, are hypermutated. These results suggest that somatic hypermutation is linked to the initiation of transcription. A model is proposed in which somatic mutation is dependent on transcription coupled DNA repair.

Animals↗
PubMed1996

A model for the mechanism of precise integration of a microinjected transgene.

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5' and 3' joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5' end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites of DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, the Escherichia coli Chi site and the meiotic recombination hotspot within the E beta gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.

Animals↗
PubMed1997

Role of mouse and human class II transgenes in susceptibility to and protection against mouse autoimmune thyroiditis.

Mouse experimental autoimmune thyroiditis (EAT), a model for Hashimoto's thyroiditis, is induced by immunizing with mouse thyroglobulin (MTg). To study the extent of H2A involvement in EAT, we introduced AaAb genes from susceptible k mice into resistant or intermediately susceptible strains which do not express H2E molecules. Thyroiditis was severe in resistant B10.M (H2(f)) mice carrying the double transgene AakAbk. Likewise, thyroid infiltration was significantly extended in intermediate B10.Q (H2(q)) mice with the same transgene. To examine the effect of H2E molecules in the presence of H2A-mediated susceptibility, we introduced an Eak transgene into E- B10.S mice to express the Ebetas molecule and observed significant reduction in EAT severity in B10.S(E+) mice. On the other hand, the presence of an Ebd transgene in B10.RQB3 (H2Aq) mice resulting in the expression of H2Ebetad molecules did not alter EAT susceptibility, suggesting a role for Eb gene polymorphism in protection against EAT. We have shown recently that the HLA-DRB1(*)0301 (DR3) transgene conferred EAT susceptibility to B10. M as well as class II-negative B10.Ab0 mice. However, we report here that the HLA-DQB1(*)0601 (DQ6b) transgene in B10.M or HLA-DQA1(*)0301/DQB1(*)0302 (DQ8) transgene in class II-negative Ab0 mice did not. These studies show the differential effects of class II molecules on EAT induction. Susceptibility can be determined when class II molecules from a single locus, H2A or HLA-DQ, are examined in transgenic mice, but the overall effect may depend upon the presence of both class II molecules H2A and H2E in mice and HLA-DQ and HLA-DR in humans.

Animals↗
PubMed1999

Introduction of the human PLTP transgene suppresses the atherogenic diet-induced increase in plasma phospholipid transfer activity in C57BL/6 mice.

The human plasma phospholipid transfer protein (PLTP) has been shown to facilitate the transfer of phospholipids between lipoproteins and convert high-density lipoproteins into larger and smaller particles in vitro. To explore the lipid transport function in vivo, transgenic C57BL/6 mice that express the human PLTP gene, driven by its natural promoter, were generated. Little difference in PLTP activity and lipoprotein lipids was observed between transgenic mice and non-transgenic control mice fed the chow diet. In response to an atherogenic high-fat, high-cholesterol, cholic acid containing diet, the PLTP activity increased significantly with time in control mice (62% in males and 34% in females after the high-fat diet for 18 weeks). In contrast, the PLTP activity did not change appreciably in the transgenic mice fed the atherogenic diet. Thus, the introduction of the human transgene suppressed the diet-induced increase in plasma PLTP activity, as evidenced by a decrease in PLTP mRNA in a variety of tissues. High-density lipoprotein levels decreased in mice fed the atherogenic diet, but there was a proportionally greater decrease in transgenic animals than in controls. After 18 weeks on the atherogenic diet, the transgenic animals had high-density lipoprotein-cholesterol and PLTP activity approximately one-half of that of control animals. Non-denaturing gradient gel electrophoresis of plasma indicated that the atherogenic diet decreased the high-density lipoprotein size distribution in control mice. However, high-density lipoprotein particle size distribution of the transgenic mice was shifted to smaller particles compared with control animals (P < 0.001). These findings suggest that PLTP activity can modulate the effects of an atherogenic diet on high-density lipoproteins.

Animals↗
PubMedDate not supplied

Accumulation of beta-amyloid fibrils in pancreas of transgenic mice.

Some forms of familial Alzheimer's disease are caused by mutations in the amyloid beta protein precursor (beta APP), and there is excellent evidence that these mutations foster amyloid deposition by increasing secretion of total amyloid beta protein (A beta) or the highly amyloidogenic A beta 1-42 form. These observations provide a powerful rationale for developing an animal model of AD by generating transgenic mice in which cerebral amyloid deposition is induced by A beta overproduction. To produce substantial A beta in vivo, we generated mice expressing the transgene of signal peptide and 99 residues of carboxyl-terminal fragment (CTF) of beta APP under control of the cytomegalovirus enhancer/chicken beta-actin promoter. The transgenic mRNA was detected in many tissues of these mice, but the levels of transgenic mRNA, CTF, and A beta did not correlate well indicating that tissue-specific posttranslational processing may play an important role in determining the amount of A beta that accumulates in various tissues. A beta was detected biochemically in brain, kidney, and pancreas with the largest amount present in pancreas. In transgenic plasma, there was a marked accumulation of human A beta 1-40 and A beta 1-42(43) to levels over 30-times those observed in normal human plasma. Thus, the transgenic mice produce and secrete considerable A beta. Despite this increase in A beta secretion and the elevated A beta in brain, immunohistochemistry revealed no consistent cerebral A beta deposition. In pancreas, however, intracellular A beta deposits were detected immunohistochemically in acinar cells and interstitial macrophages, some of which showed severe degeneration. In addition, examination of these cells by immunoelectron microscopy revealed many putative amyloid fibrils (7-12 nm) that were stained by anti-A beta antibodies. Overall, our findings indicate that tissue-specific posttranslational processing may play a pivotal role in A beta production and amyloid fibril formation in vivo. By carefully analyzing the changes that occur in the transgenic mice described here as compared to the transgenic line that has recently been shown to form extracellular amyloid plaques in brain, it may be possible to gain considerable insight into the factors that determine the location and amount of A beta that accumulates as amyloid.

Amyloid beta-Peptides↗
PubMedDate not supplied

Artifactual strain-specific signs of incipient brain amyloidosis in APP transgenic mice.

In an attempt to generate transgenic mice modeling Alzheimer-type amyloidogenesis, the COOH-terminal 103 residue human APP segment was expressed in brain regions known to be vulnerable in AD. Transfected cells overexpressing this transgene were previously shown to develop intracytoplasmic inclusions that were immunoreactive with antibodies to the APP COOH-terminus. Transgenic C57B6/SJL mice produced transgene-coded mRNA in their brains at levels up to sixfold above endogenous APP, most abundantly within cortical and hippocampal pyramidal neurons. Immunocytochemistry with anti-A beta antibodies revealed occasional structures that resembled diffuse amyloid, but which could not be detected on serial sections. Immunolabeling with antibodies to APP regions NH2-terminal to the transgene-coded domain revealed elevated immunoreactivity within perikarya and neurites in regions expressing the highest transgene and endogenous APP mRNA levels, similar to observations previously reported within vulnerable neurons in AD brain. However, subsequent breeding revealed that this phenotype segregated with the B6/SJL background rather than the transgene, thus emphasizing the importance of genetic background to observations of putative AD-type pathology in transgenic animals.

Alzheimer Disease↗
PubMedDate not supplied

Rational design of an animal model for Alzheimer's disease: introduction of multiple human genomic transgenes to reproduce AD pathology in a rodent.

A major obstacle to understanding the pathogenesis of Alzheimer's disease is the lack of easily studied animal models. Our approach is to apply transgenic methods to humanize mice and rats, employing methods that introduce large genomic transgenes, because this improves the level of transgene protein expression and the tissue specificity of expression. Our plan is to reproduce AD pathology in rodents by making them transgenic for several human proteins involved in AD. This report describes transgenic animal lines that we have produced, and summarizes our current approach and future plans. Two human genes known to be involved in AD pathology are the amyloid precursor protein (APP) and the E4 isoform of apolipoprotein E (apoE4). So far, we have produced and analyzed a transgenic line carrying the entire human APP gene cloned in a yeast artificial chromosome. We have also produced but not yet analyzed a mouse carrying the human apoE4 gene. Work is in progress to produce a transgenic line carrying a disease-causing mutation in the human APP gene. As we produce these animals, we are breeding them together, and also breeding them with a mouse line that lacks endogenous apoE, to produce an animal model carrying several human proteins whose interaction is believed to be instrumental in development of AD pathology. These transgenic animals will be useful for dissecting the biochemical and physiological steps leading to AD, and for development of therapies for disease intervention.

Alzheimer Disease↗
PubMed2005

Cardiac-specific and ligand-inducible target gene expression in transgenic mice.

Conditional transgene expression in the heart is a useful approach to explore the physiological basis of the cardiac phenotype. The present study describes the development of a binary transgenic system in which transgene expression in the mouse heart can be turned on/off by administration/withdrawal of an exogenous compound. We generated a transgenic line (alphaMHC-Glp 65) harboring a mifepristone (RU486)-controlled chimeric transcription factor (Glp 65) under the regulatory control of the cardiac-specific alpha-myosin heavy chain (alphaMHC) promoter. In the presence of RU486, Glp 65 expressed in the heart is able to bind to a target gene promoter containing four copies of the 17-mer GAL4 binding site, resulting in ligand-inducible transactivation of the target gene. We tested this system by crossing the transgenic mice, alphaMHC-Glp 65, with a transgenic line harboring human growth hormone (hGH) target gene. We observed that expression of hGH could be induced in adults as well as in the embryonic hearts of bigenic mice by RU486. The basal hGH expression was very low and the inducible level in the heart was estimated over 800-fold higher versus the basal level after 4 days of administration of RU486 at 500 microg/kg body weight per day at 2-5 months of age. The level of the transgene returned to the basal level within 7 days after withdrawal of RU486. This system can be used to control cardiac-specific expression of transgene in a time- and dose-dependent manner.

Animals↗
PubMed1999

Morphological, histochemical, immunohistochemical, and ultrastructural characterization of tumors and dysplastic and non-neoplastic lesions arising in BK virus/tat transgenic mice.

To study the role in AIDS pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Tat protein, a transactivator of viral and cellular genes, we generated transgenic mice with a recombinant DNA containing BK virus (BKV) early region and the HIV-1 tat gene, directed by its own promoter-enhancer. DNA hybridization revealed that the transgene is stably maintained in all organs of transgenic mice as a tandem insertion in a number of copies ranging from 5 to 20 per cell. In addition, tat and BKV RNA were expressed in all tissues. Transgenic mice developed three types of lesions: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. Tumors of different histotypes, such as lymphomas, adenocarcinomas of skin glands, leiomyosarcomas, skin squamous cell carcinomas, hepatomas, hepatocarcinomas, and cavernous liver hemangiomas, developed in 29% of transgenic animals. The majority of tumors were malignant, invasive, and producing metastases. Conversely, tumors of only two histotypes (lymphomas and adenocarcinomas of skin glands) appeared in control mice. Hyperplastic and dysplastic lesions were more frequent in transgenic than in control mice and involved the skin or its adnexes, the liver and the rectum, indicating multiple targets for the activity of the transgene. Pyelonephritis, frequently complicated with hydronephrosis, inflammatory eye lesions, and amyloid depositions represented the most frequent non-neoplastic lesions detected in transgenic mice. Many of the pathological findings observed in this animal model are comparable to similar lesions appearing in AIDS patients, suggesting a relevant role for Tat in the pathogenesis of such lesions during the course of AIDS.

Adenocarcinoma↗
PubMed1997

Hoxb-8 gain-of-function transgenic mice exhibit alterations in the peripheral nervous system.

To understand the developmental role of Hoxb-8, this relatively 5' Hoxb gene was ectopically expressed in embryonic regions where only more 3' Hox genes are normally expressed. Hoxb-8 coding sequences driven by a retinoic acid receptor beta2 promoter fragment were introduced in the mouse germ line by pronuclear injection. The promoter was chosen with the aim to extend rostrally the expression domain of the gene in neurectoderm and mesoderm at the time of development when Hox gene expression domains are being established. Embryos developing from DNA-injected zygotes, and from transgenic mouse lines were analyzed. Pattern alterations were observed in transgenic embryos, some of which involved the peripheral nervous system. Spinal ganglia in the mouse are first detectable around embryonic day 9.5. By day 11.5, the first of these ganglia (C1, Froriep's ganglion) has degenerated in the mouse and other amniotes. In contrast, this first ganglion did persist in the Hoxb-8 gain-of-function transgenic mice. We have started to take advantage of the phenotype of transgenic versus wild-type embryos to understand the mechanisms underlying the ontogeny and degeneration of Froriep's ganglion in wild-type mice, and the role of Hoxb-8 in C1 maintenance in transgenic embryos. The present work describes a morphological, histological and immunocytological analysis of both the degenerating and the permanent C1, and a preliminary characterization of the axonal extensions from the transgenic C1. We discuss the methodology of generating gain-of-function transgenic mice to study the genetics of pattern formation along the antero-posterior axis, and the usefulness of analyzing these particular Hoxb-8 transgenic embryos to understand some aspects of the ontogenesis and development of the upper cervical dorsal root ganglia.

Animals↗
PubMed2003

Effect of the Emu IgH enhancer on expression of a GFP reporter gene in transfected B cells and transgenic mice.

Transgenic mice were generated to identify the first B cell maturation stage showing expression of an immunoglobulin transcriptional enhancer element (Emu)-green fluorescent protein (GFP) transgene, and to check the ability of the Emu element to behave as a locus control region. Flow cytometry experiments indicated that stably transfected 18-81 cells (a murine pre-B cell line) and A20 cells (a murine IgM(+) B cell line) maintained a constant GFP expression for several months in culture. Contrasting with in vitro results, flow cytometry experiments did not highlight GFP(+) B cells in spleen and bone marrow of Emu-GFP transgenic mice and no GFP transcripts were detected by Northern blot and reverse transcriptase polymerase chain reaction analysis. In transgenic mice, the lack of GFP expression seemed related to transgene DNA methylation occurring within all organs. Our results show dramatic differences for expression of the Emu-GFP transgene in vitro and in vivo. Despite that Emu was reported to efficiently control the in vivo expression of other associated transgenes, it is not sufficient to sustain GFP expression in transgenic mice and to counteract developmental silencing programs that occur in the embryo.

Animals↗
PubMed2000

Expression of CD80 promoter in transgenic mice.

CD80 is a very potent co-stimulatory factor which is required for complete T-cell activation. Here, we use transgenic mice as a tool to map the promoter of the CD80 gene. We engineered three different CD80 promoter driven luciferase transgenes: -3084, -1073 and -215. With these transgenes, we have generated three groups of transgenic mice. Our results showed that the -3084 CD80 promoter/luciferase transgene was sufficient to confer tissue-specific expression of the CD80 gene. When the promoter sequence was deleted to -1073, the normal tissue-specific expression was lost. A brain-specific element was mapped between -1073 nt and -215 nt. This element caused up to ninefold higher expression of the CD80 promoter/luciferase in brain tissue of -1073 CD80 promoter/luciferase transgenic animals as compared to -3084 CD80 promoter/luciferase transgenic animals. In contrast to results with a cell culture system, little luciferase activity was detected in -215 CD80 promoter/luciferase transgenic animals.

Animals↗
PubMed2000

Porcine MCP gene promoter directs high level expression of human DAF (CD55) in transgenic mice.

Porcine membrane cofactor protein (pMCP), a complement regulatory protein, is widely expressed in various tissues. Particularly, it is highly expressed on vascular endothelium. The objective of this study was to investigate whether the pMCP gene promoter can induce efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55) in transgenic mice. Two fragments of the 5'-flanking region of pMCP gene (0.9 kb and 5.4 kb) connected with human DAF minigene (0.9/hDAF and 5.4/hDAF) were used to produce transgenic mice. The expression of hDAF in heart, liver, kidney, lung, pancreas, brain and testis of the transgenic mice was examined by immunohistochemical analysis. The vascular endothelia and the nerves in all organs examined were intensely stained. The staining pattern in these tissues was similar in all transgenic mice examined regardless of the length of the promoters. The surface expression levels of hDAF on peripheral red blood cells and splenocytes from a mouse carrying 5.4/hDAF hemizygously was twice the level of expression on corresponding human cells. The red blood cells and splenocytes from the transgenic mice exhibited resistance to lysis by human serum in a manner dependent upon expressed hDAF level. The hearts from the transgenic mice functioned for a significantly longer time than those from normal mice under perfusion with human serum in the Langendorff perfusion system. These results demonstrated that the pMCP gene promoter is a good candidate of the regulatory element in the transgene to produce transgenic animals for xenotransplantation.

Animals↗
PubMed2002

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Animals↗
PubMed1998

Regulation of CAT protein by ribozyme and antisense mRNA in transgenic mice.

Transgenic mouse lines were engineered to express stably antisense mRNA or antisense mRNA containing catalytic ribozyme (rbz) structures complementary to bacterial chloramphenicol acetyltransferase (CAT) gene transcripts. One transgenic line expressed antisense mRNA that specifically targeted full-length CAT coding sequences (ACAT). Another transgenic line expressed full-length antisense CAT mRNA which was modified by mutagensis to include four rbz cassettes (rbz-ACAT) in order to compare antisense versus antisense-rbz function in vivo. Preliminary data were also collected from a transgenic mouse line expressing antisense mRNA targeting 72% of the 5' region of CAT coding sequences (5' ACAT). All constructs contained similar control elements in their design. Promoter elements were derived from the bovine alpha s1-casein gene, while the small t intron and 3' control sequences were derived from SV40. The ability of these various constructs to down-regulate CAT protein levels was compared by analysis of CAT protein production in lactating double-hemizygous transgenic female mice. Every double-hemizygous mouse analysed expressed mRNA from the alpha s1-casein-CAT construct (Clarke et al., 1994) and equivalent levels of mRNA from one of the three antisense constructs. Transgenic mouse lines expressing both ACAT and CAT mRNA down-regulated CAT protein levels by 90% of that found in the CAT only transgenic population. Similarly, double-hemizygous transgenic lines expressing both rbz-ACAT and CAT mRNA regulated CAT protein levels by 87%. Preliminary data suggests that expression of mRNA from 5' ACAT/CAT double-hemizygote mice allowed approximately 67% down-regulation of normal CAT protein levels. We conclude that incorporation of multiple ribozymes within the full-length antisense CAT construct does not enhance the effectiveness of antisense mRNA in the down-regulation of CAT protein production in our system.

Animals↗
PubMed1998

Tissue-specific expression of human salivary mucin gene, MUC7, in transgenic mice.

The MUC7 gene encodes the protein core of the low molecular weight human salivary mucin (MG2, mucin glycoprotein 2) and is expressed in a tissue-specific manner in salivary glands. The purpose of this study was to examine MUC7 expression by transgenic mouse technology. A 16 kb DNA fragment, containing the MUC7 gene (10 kb) and 3 kb of the upstream and 3 kb of the downstream sequences, was used to generate transgenic mice. We have identified five transgenic founder mice which were propagated as individual transgenic lines and analysed. Tissues of transgenic offspring from each line were analysed by RT-PCR to determine the sites of the MUC7 expression. The results indicated that only line 3 and line 5 expressed the MUC7 gene in salivary glands. The level of MUC7 expression in selected tissues was then determined by northern blot analyses. The results showed that line 3 mice contained high levels of MUC7 transcripts in the sublingual glands of both males and females and indicated low levels of MUC7 transcripts in the submandibular glands of females. No MUC7 expression was detected in this line by northern blot analysis in any other tissue tested. On the other hand, no expression of MUC7 was detected in any tissues of line 5 mice examined by northern blot analysis. A Southern blot analysis of human and mouse genomic DNA demonstrated multiple copies of the MUC7 transgene in line 3 and a single copy in line 5. Collectively, these results indicate that the regulatory sequences required for the tissue-specific expression of MUC7 are within the MUC7 transgene. However, the sequences necessary for expression comparable to that of MUC7 in human salivary glands may be missing from this construct. Western blot analysis of protein extracts from different tissues of transgenic mice line 3 showed that MUC7 gene product was produced in the submandibular-sublingual gland complex of both male and female mice and not in the other tissues examined.

Animals↗
PubMed2000

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos.

The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin. The experiments reported here were designed to determine if the inclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identification using a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryos were microinjected with GFP reporter gene under the control of three different promoters and flanked or not by three different MAR elements. Transgene expression profiles were followed by direct visualization of GFP in cultured microinjected embryos. Embryos at different developmental stages were classified according to their GFP expression and groups with the same expression pattern were transferred into oviducts of pseudopregnant female mice. Fetuses were collected between days 12-15, and their genomic DNA was purified and analyzed to detect transgene integration. We did not find any statistically significant difference between the percentage of transgenic fetuses produced from GFP-positive or GFP-negative embryos transferred at 4-cell, morula, or blastocyst stage. However, when MAR elements were included in the construct, we found that GFP-positive embryos transferred at the 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, but MAR sequences did not completely eliminate false positives.

Animals↗
PubMed2003

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Alleles↗