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PubMed1993

Thymus-independent positive and negative selection of T cells expressing a major histocompatibility complex class I restricted transgenic T cell receptor alpha/beta in the intestinal epithelium.

We demonstrate that in the mouse intestinal epithelium the selection of T lymphocytes expressing a transgenic T cell receptor alpha/beta (TCR-alpha/beta) specific for male antigen (H-Y) in the context of H-2Db depends on the differential expression of H-Y and H-2Db in situ. In H-2Db transgenic males, there is no reduction in the number of intestinal intraepithelial lymphocytes (IEL), and the four main subsets of IEL expressing TCR-alpha/beta, defined by the differential expression of CD4, CD8 alpha, and CD8 beta, are present. Moreover, the level of expression of CD8 alpha and CD8 beta on CD8+ IEL subsets is unaltered. The frequency of CD8 alpha+ IEL expressing CD8 beta, in H-2Db male mice, however, is significantly decreased and these cells do not express the transgenic TCR. In contrast, virtually all CD8 alpha+beta- IEL in the same animals express the transgenic TCR. Still, these potentially autoreactive cells are refractile to H-Y/H-2Db stimulation in vitro. Both H-2Db and H-2Dd transgenic females contain high frequencies of cells expressing the transgenic TCR among CD8 alpha+beta- and CD8 alpha+beta+ IEL. However, two possibly related phenotypic features are peculiar to H-2Db female mice. The frequency of CD8 alpha+ IEL expressing CD8 beta is increased in these mice and, while in H-2Dd females the level of the transgenic TCR alpha chain expressed on CD8 alpha+beta+ IEL is uniformly low, some of the CD8 alpha+beta+ IEL in H-2Db females express a high level of both transgenic TCR chains. It is important to note, the ability of CD8 alpha+beta+ IEL to respond to H-Y/H-2Db stimulation in vitro is restricted to those coexpressing a high level of both transgenic TCR chains. The analysis of athymic radiation chimeras using adult thymectomized recipients of distinct H-Y/H-2 haplotypes, reconstituted with bone marrow from H-2Db transgenic females, demonstrates that all IEL subsets present in unmanipulated transgenic animals develop in the absence of a thymus. These IEL are phenotypically identical to those found in unmanipulated transgenic animals sharing the H-Y/H-2 haplotype of athymic recipients. Taken together, these results demonstrate that in the absence of male antigen, expression of H-2Db in the intestinal epithelium results in the positive selection of functional IEL specific for male antigen, in situ. When both H-Y and H-2Db are expressed in the intestinal epithelium, CD8 alpha+beta+ IEL expressing the transgenic TCR are negatively selected, while the frequency of nonfunctional CD8 alpha+beta- IEL expressing the transgenic CR is increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Transgene integration in hair follicles and peripheral blood cells measured by in vitro DNA amplification and fluorescence in situ hybridization.

Screening of animals to detect the presence of integrated DNA sequences is an essential component of transgenic mouse generation. Rapid and sensitive detection techniques to facilitate identification of transgenic animals for biological studies or subsequent breeding programs are desirable. Most transgenics are generated on F1 backgrounds, thus determination of the histocompatibility status of neonates provides important diagnostic information for establishing congenic colonies. We describe the application of two assays, in vitro DNA amplification using the polymerase chain reaction (PCR) and fluorescence in situ hybridization with biotinylated DNA probes, to facilitate rapid detection of transgenes and their chromosomal integration patterns in young mice. A noninvasive PCR-based assay to detect the transgene in DNA contained in detergent-extracted hair follicles was developed for rapid screening. A total of 147 mice derived from F2, F3, and F4 generations of C57BL x F1 (globin transgenics) were assayed to determine whether they carried a globin transgene. Characterization of animals by PCR-based amplification of the transgene was compared with that obtained using standard Southern analysis of DNA extracted from tails. Categorization of animals as positive (carrying the transgene) or negative using PCR was performed successfully in the initial assay with 95% of the animals. Fluorescence in situ hybridization with a DNA probe showing homology with a portion of the transgene was performed on metaphase and interphase cells to determine the integration pattern of the transgene. Our data showed that the transgene was integrated in a single chromosome. These techniques should facilitate rapid identification of transgenic animals and characterization of the genomic transgene integration patterns.

Animals↗

PubMed1994

Human insulin-like growth factor-binding protein-1 (hIGFBP-1) in transgenic mice: characterization and insights into the regulation of IGFBP-1 expression.

Three hemizygous transgenic (Tg) mouse lines were generated with a fusion gene composed of the mouse metallothionein promoter (mMT-I) and a full-length human insulin-like growth factor binding protein-1 (hIGFBP-1) complementary DNA that was truncated in its 3'-untranslated region. Despite high serum hIGFBP-1 levels (120-2570 micrograms/liter) before puberty in two of these lines, no significant alterations were observed in somatic growth, nor were marked alterations noted in fasting or random serum glucose or in the response of young adult Tg mice to ip glucose. The transgene was expressed in a number of tissues from each line, but liver was a significant site of transgene expression in only one line. Unexpectedly, liver hIGFBP-1 messenger RNA (mRNA) expression in this line was regulated in fashion similar to the native liver IGFBP-1 mRNA: 1) its abundance waned with advancing postnatal age and became minimal in early adult life, despite continuous zinc supplementation to stimulate its transcription; and 2) fasting increased its abundance 3- to 4.3-fold. The decline in transgene expression with aging was not due to a deletion, rearrangement, or a change in the methylation of liver transgene DNA. Transcriptional mechanisms also were not likely to account for the observed regulation of the transgene mRNA, because liver expression of the mMT-I gene, which shares identical genomic 5'-regulatory elements with the transgene, was not similarly altered by aging or fasting. Because cycloheximide (CHX) treatment of cultured rat H4IIE cells has been shown to prolong IGFBP-1 mRNA half-life while decreasing its transcription, Tg mice were treated with CHX to test the possibility that instability of the liver transgene mRNA influenced its abundance. After CHX and under conditions of chronic zinc supplementation, liver transgene mRNA abundance increased in parallel with that of the native IGFBP-1 mRNA. Although CHX is known to activate mMT-I transcription by mechanisms involving the 5'-regulatory regions contained in the transgene, CHX-induced transcription only in part accounted for the increase in liver transgene mRNA, because CHX induced an earlier and greater increase in liver transgene mRNA than in mMT-I mRNA. Taken together, these data indicate that both transgene and native IGFBP-1 liver mRNA are regulated by factors that alter mRNA stability. The finding that native liver IGFBP-1 mRNA abundance is influenced by transgene expression further supports the concept that both mRNAs share some common mechanisms of regulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2000

Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant.

PURPOSE: Lim2 is the gene encoding the ocular lens-specific intrinsic membrane protein MP19. We previously reported finding a single nonconservative G->T transversion in exon two of the Lim2 gene. This mutation was linked to the cataract in the To3 (Total opacity number 3) mouse mutant, confirming Lim2 as an ideal candidate gene for the To3 cataract. The aim of the present study was to substantiate a causative relationship between the mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. To this end a Lim2To3 transgene cassette was engineered and introduced into fertilized normal mouse embryos to test its ability to induce cataractogenic lens development. METHODS: A Lim2 genomic clone was isolated and purified from a murine 129/SvJ genomic library. A restriction endonuclease map of the gene was generated using classical Southern techniques. The murine Lim2 promoter was characterized by transfecting primary chicken lens epithelial cells with Lim2 promoter-CAT reporter constructs and assaying promoter activity and specificity. This genomic clone was then used in conjunction with PCR to generate a Lim2To3 transgene cassette. After sequencing of the PCR engineered portion, the Lim2To3 transgene was then used to generate Lim2To3 transgenic mice via pronuclear injection. Founder mice and their offspring from outcrosses and intercrosses were characterized by ophthalmic examination, PCR and Southern DNA analysis, RT-PCR mRNA analysis, and histology of lens sections. RESULTS: Two mice, from independent microinjections, were identified as positive for presence of the Lim2To3 transgene cassette as well as presence of bilateral congenital cataracts and reduced eye size and mass. One of these founders was incapable of germline transmission of the transgene to offspring and was not characterized further. The other was capable of germline transmission and was characterized as described above. PCR DNA analysis revealed a perfect concordance between presence of the Lim2To3 transgene cassette and congenital cataract in offspring of this founder. Transgenic hemizygotes exhibited cataract and a reduction in eye and lens size and mass, while transgenic "homozygotes" presented with a more severe cataract and microphthalmic reduction in eye and lens size and mass. Southern analysis revealed approximately 2 copies of the transgene cassette integrated into a single chromosomal site in the founder and all hemizygous offspring. RT-PCR analysis revealed a very low ratio of Lim2To3 transgenic mRNA compared to endogenous normal Lim2. Finally, histology revealed that lens development was abnormal in mutant transgenic animals by embryonic day E15. By E19, just prior to birth, gross disorganization of secondary fibers was observed in mutants. CONCLUSIONS: These transgenic experiments firmly establish a causative relationship between the previously identified mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. The low levels of mutant mRNA produced by the transgene cassette as compared to endogenous levels of normal Lim2 mRNA provides evidence that this dominant mutation results in a mutant MP19 protein with altered function rather than simply loss of function.

Animals↗

PubMed1994

Overexpression of transforming growth factor-alpha causes liver enlargement and increased hepatocyte proliferation in transgenic mice.

Transforming growth factor-alpha (TGF-alpha) expression is associated with hepatocyte DNA replication both in vivo and in culture. Our previous work using TGF-alpha transgenic mice showed that constitutive overexpression of this growth factor in the liver causes hepatic tumors in 75 to 80% of the animals at 12 to 15 months of age. To understand the cellular events by which TGF-alpha overexpression leads to abnormal liver growth, we examined hepatocyte proliferative activity in young and old TGF-alpha transgenic mice and hepatocyte ploidy in normal, dysplastic, and neoplastic livers of these animals. At 4 weeks of age, transgenic mice had higher liver weights and liver weight/body weight ratios than non-transgenic mice of the same age and hepatocyte proliferative activity, measured by 3H-thymidine incorporation after 3- and 7-day infusion, proliferating cell nuclear antigen staining, and mitotic index determination, was 2 to 3 times higher than in controls. In both transgenic and non-transgenic mice hepatocyte proliferation declined with age but the decrease was much more pronounced in control animals, so that at 8 months of age, hepatocyte replication was 8 to 10 times higher in transgenic animals. Surprisingly, however, transgenic and non-transgenic mice at this age had similar liver weight/body weight ratios. Labeling studies done in 3-month-old animals revealed that hepatocyte turnover was much faster in transgenic than in control animals, suggesting that a homeostatic compensatory mechanism involving cell death tended to restore normal liver weight/body weight ratios in older transgenic mice. Ploidy analyses showed that at 4 weeks of age transgenic mice had a higher proportion of diploid and tetraploid hepatocytes and that the hepatocellular tumors which developed in TGF-alpha transgenic mice at 13 months of age contained a higher fraction of diploid hepatocytes than that present in adjacent tissue or in dysplastic livers. The results demonstrate that constitutive overexpression of TGF-alpha causes increased hepatocyte proliferation and liver enlargement in young animals and is associated with a delay in the establishment of hepatic polyploidy. These findings as well as the response of transgenic mice to partial hepatectomy show that constitutive overexpression of TGF-alpha initially caused increased but regulated hepatocyte proliferation which in older animals was compensated in part by a faster cell turnover. At 8 to 10 months of age, proliferative activity may become constitutive in some TGF-alpha expressing hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1996

Skeletal muscle growth of oMTla-oGH transgenic mice.

Forty-eight transgenic mice carrying an ovine metallothionein 1a-ovine growth hormone (oMTla-oGH) transgene and 48 littermate control mice were used to investigate the effect of GH transgene on the growth and biochemical characteristics of skeletal muscle. Transgene expression was initiated in the transgenic mice by the addition of zinc sulfate to the water at 21 d of age; control mice were also supplemented with zinc sulfate. These mice were maintained on zinc sulfate until 84 d of age. Groups of mice (16 controls, 16 transgenics) were killed at 21, 42 and 84 d of age and muscles from the hind leg were dissected, weighed and analyzed. At 84 d, male transgenics were 32% heavier than controls, while female transgenics were 47% heavier. Transgenic mice of both sexes had smaller (p < 0.01) muscles than controls at weaning (21 d). In spite of significantly heavier body weights of transgenic mice at 84 d of age, there were no significant differences in muscle weights. This was due to a significantly lower (p < 0.01) proportion of muscle, expressed as percentages of body weights, in transgenic mice compared with controls. Higher DNA and RNA concentrations at 42 d of age and elevated cathepsins C and H activities at 42 and 84 d of age indicate that muscle protein metabolism is more active in transgenic mice, which are growing at a greater rate than controls from weaning to 84 d of age. The fact that oMTla-oGH transgenic mice inherently have a lower proportion of muscle, compared to controls and that this proportion does not change in spite of transgene activation and 30 fold increase in plasma GH levels, suggests the hypothesis that muscle growth may be controlled by locally produced IGFs, which are essentially independent of circulating GH concentrations.

Animals↗

PubMed2007

The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants.

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a beta-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chi-MARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chi-MARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.

Animals↗

PubMed2003

Supplements of transgenic malt or grain containing (1,3-1,4)-beta-glucanase increase the nutritive value of barley-based broiler diets to that of maize.

1. A diet with addition to normal barley of malt from transgenic barley expressing a protein engineered, thermotolerant Bacillus (1,3-1,4)-beta-glucanase during germination has previously been demonstrated to provide a broiler chicken weight gain comparable to maize diets. It also reduced dramatically the number of birds with adhering sticky droppings, but did not entirely eliminate sticky droppings. One of the objectives of the broiler chicken trials reported here was to determine if higher concentrations of transgenic malt could alleviate the sticky droppings. 2. Another aim was to investigate the feasibility of using mature transgenic grain containing the thermotolerant (1,3-1,4)-beta-glucanase as feed addition and to compare diets containing transgenic grain to a diet with the recommended amount of a commercial beta-glucanase-based product. 3. Inclusion of 75 or 151 g/kg transgenic malt containing 4.7 or 98 mg/kg thermotolerant (1,3-1,4)-beta-glucanase with 545 or 469 g/kg non-transgenic barley instead of maize yielded a weight gain in Cornish Cross broiler chickens indistinguishable from presently used maize diets. The gene encoding the enzyme is expressed in the aleurone with a barley alpha-amylase gene promoter and the enzyme is synthesised with a signal peptide for secretion into the endosperm of the malting grain. 4. Equal weight gain was achieved, when the feed included 39 g/kg transgenic barley grain [containing 66 mg/kg thermotolerant (1,3-1,4)-beta-glucanase] and 581 g/kg non-transgenic barley instead of maize. In this case, the gene encoding the enzyme has been expressed with the D-hordein gene (Hor3-1) promoter during grain maturation. The enzyme is synthesised as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeted into the storage vacuoles. Deposition of the enzyme in the prolamin storage protein bodies of the endosperm protects it from degradation during the programmed cell death of the endosperm in the final stages of grain maturation and provides extraordinary heat stability. The large amount of highly active (1,3-1,4)-beta-glucanase in the mature grain allowed the reduction of the transgenic grain ingredient to 0.2 g/kg diet, thus making the ingredient comparable to that of the trace minerals added to standard diets. 5. A direct comparison using transgenic grain supplement at the level of 1 g/kg of feed with the standard recommended addition of the commercial enzyme preparation Avizyme 1100 at 1 g/kg yielded equal weight gain, feed consumption and feed efficiency in birds fed a barley-based diet. 6. The production of sticky droppings characteristic of broilers fed on barley diets was avoided with all 9 experimental diets and reduced to the level observed with a standard maize diet by supplementation with transgenic barley. 7. The excellent growth and normal survival of the 400 broilers tested on barley diets supplemented with transgenic grain or malt showed the grain and malt not to be toxic. 8. The barley feed with added transgenic grain or malt containing thermotolerant (1,3-1,4)-beta-glucanase provides an environmentally friendly alternative to enzyme additives, as it uses photosynthetic energy for production of the enzyme in the grain and thus avoids use of non-renewable energy for fermentation. The deposition of the enzyme in the protein bodies of the grain in the field makes coating procedures for stabilisation of enzyme activity superfluous. 9. Barley feed with the small amount of transgenic grain as additive to normal barley provides an alternative for broiler feed in areas where grain maize cannot be grown for climatic reasons or because of unsuitable soil and thus has to be imported.

Animal Feed↗

PubMed2000

Transgene translatability increases effectiveness of replicase-mediated resistance to cucumber mosaic virus.

Transgenic tobacco plants expressing an altered form of the 2a replicase gene from the Fny strain of Cucumber mosaic virus (CMV) exhibit suppressed virus replication and restricted virus movement when inoculated mechanically or by aphid vectors. Additional transformants have been generated which contain replicase gene constructs designed to determine the role(s) of transgene mRNA and/or protein in resistance. Resistance to systemic disease caused by CMV, as well as delayed infection, was observed in several lines of transgenic plants which were capable of expressing either full-length or truncated replicase proteins. In contrast, among plants which contained nontranslatable transgene constructs, only one of 61 lines examined exhibited delays or resistance. Once infected, plants never recovered, regardless of transgene translatability. Transgenic plants exhibiting a range of resistance levels were examined for transgene copy number, mRNA and protein levels. Although ribonuclease protection assays demonstrated that transgene mRNA levels were very low, resistant lines had consistently more steady-state transgene mRNA than susceptible lines. Furthermore, chlorotic or necrotic local lesions developed on the inoculated leaves of transgenic lines containing translatable transgenes, but not on inoculated leaves of lines containing nontranslatable transgenes. These results demonstrate that translatability of the transgene and possibly expression of the transgene protein itself facilitates replicase-mediated resistance to CMV in tobacco.

Amino Acid Sequence↗

PubMed2003

[Expression of Mortierella isabellina delta6-fatty acid desaturase gene in gamma-linolenic acid production in transgenic tobacco].

Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta6.9.12.15) respectively have 19.7% and 3.5% of the total fatty acids in the transgenic plant. The presence of both GLA and OTA indicates that the delta6-fatty acid desaturase used both linoleic acid and a-linolenic acid (ALA, C18:3delta6.9.12.15) as substrates, and this may be responsible for the decrease in ALA observed in the transgenic line. That was the first report about the expression of M. isabellina delta6-fatty acid desaturase gene in tobacco. All results mentioned above have laid the foundation of the thorough studying on an breeding transgenic oilseeds containing GLA to change the fatty acid composition of conventional oilseeds, it is significant to study on regulation mechanism of fatty acid desaturase.

Agrobacterium tumefaciens↗

PubMed1995

A postimplantation lethal mutation induced by transgene insertion on mouse chromosome 8.

We have produced three lines of transgenic mice that contain additional copies of the mouse phosphoglycerate kinase 1 (Pgk1) gene. Two of these lines, 94-A and 94-K, which are descendants of a common founder, did not produce liveborn progeny carrying two copies of these transgenes (i.e., A/A, K/K, or A/K). Genotyping of midgestation embryos showed that A/K embryos are dead by Embryonic Day 10. Comparison of the level of transgene expression in the three transgenic lines ruled out PGK1 toxicity as the cause of death of A/A, A/K, and K/K embryos. The death of A/A, K/K, and A/K transgenic mice was therefore attributed to an insertional mutation disrupting a gene or genes essential for normal embryogenesis. Analysis of the structure of the 94-A and 94-K transgenes indicated that they differ in the number of tandem repeats and in the positions of the transgene-cellular DNA junctions. To determine if the two transgenes represent a single integration followed by a rearrangement or two independent integration events, we cloned the endogenous sequences surrounding the 94-A and 94-K transgene insertion sites. Restriction analysis of the isolated genomic clones indicated that the endogenous sequences abutting the 3' ends of the 94-A and 94-K transgenes are separated by less than 20 kb, providing strong support for the single integration model. Further analysis indicated that the 94-A transgene is associated with a deletion of at least 18 kb and is located in the vicinity of a widely transcribed endogenous gene. Chromosomal mapping of the endogenous sequences flanking the 94-A and 94-K transgene insertions using mouse-hamster somatic cell hybrids and a (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei backcross panel allowed us to assign the 94-A(K) transgene insertion to the subcentral region of mouse chromosome 8.

Animals↗

PubMed2005

An efficient system for tissue-specific overexpression of transgenes in podocytes in vivo.

The utility of promoter fragments isolated from the 5'-flanking region of endogenous mammalian genes to drive transgene expression in vivo is often limited by low expression levels. In this study, a bigenic system was established that allows constitutive overexpression of transgenes in a tissue-specific fashion in transgenic mice in a time- and cost-effective fashion. A modified floxed expression vector was constructed [CMVflox-enhanced green fluorescent protein (eGFP)], in which a lacZ cassette (beta-galactosidase) flanked by lox sites was placed between a CMV-promoter and the transgene of interest (eGFP). Before Cre recombination, expression of eGFP was effectively prevented by the interposed floxed lacZ cassette, whereas beta-galactosidase was strongly expressed in transiently transfected cells. Transcription of the gene of interest (eGFP) could be irreversibly activated by cotransfection with Cre recombinase. Mice transgenic for CMVflox-eGFP were generated by pronuclear injection. A rapid assay was developed to identify transgenic founders with active transgene expression by measuring transgene activity (beta-galactosidase) in tail biopsies. Transgene activity in tails correlated with transgene expression in most other tissues tested including podocytes within the kidney. To activate expression of the gene of interest in a tissue-specific fashion, founder mice were mated to the Cre mouse line 2.5P-Cre previously shown to mediate 100% Cre recombination exclusively in podocytes (Moeller MJ, Sanden SK, Soofi A, Wiggins RC, and Holzman LB. Genesis 35: 39-42, 2003). In doubly transgenic offspring, high-level eGFP expression resulting from Cre excision of the interposed lacZ cassette was detected in four of seven CMVflox-eGFP founder lines. This approach should also circumvent common limitations arising from lethality or transgene silencing as a consequence of transgene overexpression.

Animals↗

PubMed2000

Possible role of cytotoxic T cells in acute liver injury in hepatitis C virus cDNA transgenic mice mediated by Cre/loxP system.

A line of hepatitis C virus (HCV) transgenic mice was established previously that was mediated by Cre/loxP system using HCV cDNA, including core, E1, E2 and NS2 genes. Intravenous infection of a recombinant adenovirus that expresses Cre DNA recombinase (AxCANCre) induced HCV structural protein expression in the liver of transgenic mice. HCV core protein production and transgene recombination in the mouse liver were serially evaluated after AxCANCre infusion. Core proteins were expressed efficiently and transgene was almost completely recombined in the liver of mice after 3 days and then the levels of both core protein production and transgene recombination decreased continuously for 28 days. However, 30.6% of the transgene recombination remained at 28 days and only 2.7% of core production remained at 28 days after infection. Compared with nontransgenic controls, the serum alanine aminotransferase levels in transgenic mice were significantly higher 10, 14, and 21 days after adenovirus infection. Histological scoring also indicated severe pathological changes in the liver of transgenic mice after adenovirus infection. AxCANCre infusion increased CD8+ lymphocyte infiltration into the liver of transgenic mice compared with that of non-transgenic controls. Furthermore, cytotoxic T lymphocytes (CTLs) isolated from transgenic mice during liver injury were specific for the HCV proteins. These results suggest that HCV structural proteins expressed in the liver of transgenic mice enhanced liver injury. HCV-specific CTLs may be to enhance hepatitis. Thus, the present HCV transgenic mouse model provides a useful model of liver injury due to HCV, and the host immune response may play a pivotal role(s) in the pathogenesis of HCV.

Adenoviridae↗

PubMed1992

The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.

Animals↗

PubMedDate not supplied

Increased glial fibrillary acidic protein (GFAP) levels in the brains of transgenic mice expressing the bovine growth hormone (bGH) gene.

Transgenic mice, expressing the gene for bovine growth hormone (bGH), exhibit increased body size, reduced reproductive capacity, and high basal levels of several hormones including corticosterone. Their shortened life span may be indicative of accelerated aging. As prominent astrogliosis of the CNS accompanies aging in rodents, bGH transgenic mice were examined for astrogliosis, as quantified by an ELISA for the astrocyte-localized protein, glial fibrillary acidic protein (GFAP). Transgenic mice were produced by mating C57BL/6 x C3H F1 hybrid females with male descendants of animals produced by microinjection of fertilized eggs with phosphoenolpyruvate carboxykinase (PEPCK)/bGH-hybrid gene. Transgenic mice (approximately 3.5 and approximately 12 months of age) weighed significantly more than same age or older (approximately 20 month) controls. Most of their internal organs, including the heart, kidneys, adrenals, liver, and spleen, were also heavier. In contrast, the thymus was heavier only in the younger transgenic mice. Serum corticosterone was highest in the older transgenic mice. A small but significant increase in whole brain, cortex, and cerebellar weight, relative to controls and the older transgenic mice, was found in the younger transgenic mice. Control mice exhibited large, significant age-related increases in GFAP. Increases of 35, 70, 68, 89, 79, and 95% for cortex, cerebellum, striatum, hippocampus, midbrain, and brain stem, respectively, were found when comparing the oldest (approximately 20 months) control mice to the youngest (approximately 3.5 months). In contrast, in the olfactory bulbs and the hypothalamus there were no age-related changes in the levels of GFAP in control mice. Transgenic mice (approximately 3.5 months) had significantly elevated GFAP levels relative to the same-age controls in all brain areas examined. In some brain areas, the GFAP levels found in the younger transgenic mice were equivalent to those found in the oldest controls. No differences between controls and transgenics were found in tyrosine hydroxylase protein levels of striatum or hypothalamus. The elevated GFAP levels of transgenic mice may reflect increased neural damage due to accelerated aging processes or damage associated with high circulating levels of bGH or corticosterone. Alternatively, the increased expression of GFAP in the transgenic mice may reflect altered regulation of GFAP rather than an increase signaled by neural damage.

Aging↗

PubMed1999

Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis.

Previous attempts to establish transgenic mouse models to study the functions of transforming growth factor beta1 (TGFbeta1) in the skin revealed controversial roles for TGFbeta1 in epidermal growth (inhibition vs. stimulation) and resulted in neonatal lethality in one instance. To establish a viable transgenic model for studying functions of TGFbeta1 in the skin, we have now developed transgenic mice, which allow focal induction of the TGFbeta1 transgene in the epidermis at different expression levels and at different developmental stages. This system, termed "gene-switch," consists of two transgenic lines. The mouse loricrin vector targets the GLVPc transactivator (a fusion molecule of the truncated progesterone receptor and the GAL4 DNA binding domain), and a thymidine kinase promoter drives the TGFbeta1 target gene with GAL4 binding sites upstream of the promoter. These two transgenic lines were mated to generate bigenic mice, and TGFbeta1 transgene expression was controlled by topical application of an antiprogestin. On epidermal-specific induction of the TGFbeta1 transgene, the BrdUrd labeling index in the transgenic epidermis decreased 6-fold compared with controls. Induction of the TGFbeta1 transgene expression also caused epidermal resistance to phorbol 12-myristate 13-acetate-induced hyperplasia, with a reduction in both epidermal thickness and BrdUrd labeling compared with those in controls. In addition, TGFbeta1 transgene expression induced an increase in angiogenesis in the dermis. Given that the TGFbeta1 transgene can affect both the epidermis and dermis, this transgenic model will provide a useful tool for studying roles of TGFbeta1 in wound-healing and skin carcinogenesis in the future.

Animals↗

PubMed1991

Fertility of transgenic female mice expressing bovine growth hormone or human growth hormone variant genes.

Although growth hormone (GH) exerts various direct and indirect stimulatory effects on gonadal development and function, excessive levels of GH in acromegalic patients and in transgenic animals are often associated with reproductive disorders. We have examined reproductive performance of transgenic female mice expressing the following hybrid genes: mouse metallothionein-1 (MT)/human placental GH variant (hGH.V), MT/bovine GH(bGH), and phosphoenolpyruvate carboxykinase (PEPCK)/bGH. This allowed us to evaluate the effects of chronic GH excess in three animal models and to obtain some information on the significance of the lactogenic activity of the foreign GH (hGH.V vs. bGH) and on the developmental stage of transgene expression (MT vs. PEPCK). Transgenic animals from each line had elevated plasma insulin-like growth factor-I levels and greatly increased adult body weight. Plasma bGH levels were significantly higher in PEPCK/bGH than in MT/bGH transgenic mice. Approximately 20% of transgenic MT/hGH.V and MT/bGH females and over 60% of transgenic PEPCK/bGH females were infertile. Transgenic females that did reproduce ovulated either a normal or increased number of eggs but exhibited a variety of reproductive disorders including increased interval between pairing with a male and conception, increased interval between litters, reduced number of litters, reduced fetal growth, increased pre- and postnatal mortality, and alterations in sex ratio. Among adult offspring of these females, the proportion of transgenic animals was significantly less than the expected 50%. While some characteristics (e.g., fetal crown-rump length and weight on Day 14 of pregnancy) were affected to a comparable extent in transgenic females from all three lines, MT/hGH.V and PEPCK/bGH females were, in general, more severely affected than the MT/bGH animals. Sterility of PEPCK/bGH females appeared to be due to luteal failure since treatment with progesterone led to pregnancy. Greatly increased intervals between successive litters appeared to be due to failure to mate during postpartum estrus and to sterile matings during this period. Reduced fetal size and weight may have been due to chronic glucocorticoid excess because comparable changes could be induced in normal females by injections of dexamethasone during pregnancy, and plasma corticosterone levels were previously shown to be elevated in transgenic mice from each of these lines. Comparison of these results with data obtained from matings of normal female mice to transgenic males from the same lines suggests that reduced fetal growth is due primarily to maternal genotype, while reduced "transmission" of the hybrid genes is not, and presumably reflects increased mortality of transgenic progeny at various stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1999

Human growth hormone transgene expression increases the biomechanical structural properties of mouse vertebrae.

STUDY DESIGN: Caudal vertebrae were obtained from male and female mice from two transgenic lines expressing an erythroid-specific human growth hormone transgene construct, and gender-matched, age-matched, non-transgenic control mice. OBJECTIVE: To characterize the effect of human growth hormone transgene expression on the biomechanical structural properties of caudal vertebrae in compression. SUMMARY OF BACKGROUND DATA: An increase in trabecular and cortical bone deposition caused by erythroid-specific human growth hormone transgene expression was demonstrated previously. METHODS: Compression tests were performed on individual caudal vertebrae (Ca4, Ca5, Ca6) obtained from male and female mice from two transgenic lines (TG420 and TG450) and nontransgenic control mice. Two age groups were evaluated: 12 weeks old and 6 months old. The data were used to obtain axial stiffness, maximum load, and energy to failure. RESULTS: Vertebrae from male TG420 transgenic mice produced significantly larger values for maximum load, energy to failure, and axial stiffness at both 12 weeks and 6 months in comparison with their age-matched non-transgenic male controls. Vertebrae from female TG420 transgenic mice produced similar responses at 6 months. Vertebrae from male TG450 transgenic mice showed significant increases in maximum load and energy to failure at 6 months. In general, the biomechanical properties of vertebrae were significantly larger in the 6-month age group than in the 12-week age group, and this increase was significantly greater in the transgenic mice than in the gender-matched control mice during the same time span. This process was also influenced by transgenic genotype and gender. CONCLUSIONS: Erythroid-specific production of human growth hormone in transgenic mice resulted in significant increases in biomechanical properties of their caudal vertebrae in compression. The changes in the biomechanical properties were influenced by genotype, age, and gender.

Aging↗