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PubMed2004

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR.

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.

Blotting, Southern↗
PubMed2002

Rapid localization of transgenes in mouse chromosomes with a combined Spectral Karyotyping/FISH technique.

We explored the feasibility of combined Spectral Karyotyping (SKY) and Fluorescence In Situ Hybridization (FISH) as means to rapidly map a chromosomally integrated renin/green fluorescent protein (GFP) fusion gene construct (Ren-GFP) in the transgenic mouse, Tg(Ren-GFP)1Kwg. A sequential hybridization with SKY probes followed by FISH gave consistently satisfactory results, demonstrating that multiple copies of the Ren-GFP transgene in this transgenic mouse line are integrated into a single chromosomal site of Chromosome (Chr) 4, most probably in the juxta-centromeric euchromatic region consisting of the A2-A3 domain. Chr 4 as a sole carrier of the transgene also was confirmed by co-hybridization to p1 BAC clone DNA containing telomeric sequences specific for mouse Chr 4 and the Ren-GFP construct in pGEM4Z vector. The hemizygosity of the Ren-GFP transgene is maintained not only in bone marrow cells, but also in lung cells proliferating in vitro, indicating that stable integration of the Ren-GFP transgene into chromosomal DNA was established at a very early embryonic stage. We conclude that the SKY/FISH technique is a reliable and facile method for establishing the integration site of a transgene. As such, this protocol has obvious advantages over traditional backcross methods in terms of time, cost and labor for determining the chromosomal location of transgenes.

Animals↗
PubMed2002

Studies on the immunogenicity of hCEA in a transgenic mouse model.

BACKGROUND AND AIMS: Immunization protocols in mice have shown that the tumor-associated antigen hCEA could be a target for active immunization; however, human CEA is foreign to mice. Success may depend in part on a simple anti-xenoresponse. Using hCEA-transfected syngeneic tumor cells in hCEA-transgenic mice should bypass this problem and allow testing for new vaccination strategies. MATERIALS AND METHODS: We established a hCEA transgenic model of the haplotype H2(d), which may differ from other haplotypes in cytokine production and in effectiveness of antigen presentation, and tested two vaccination protocols in wild-type and transgenic mice. RESULTS: Syngeneic wild-type mice built up an immune response with high antibody titers; only 65% of animals developed solid tumors after tumor challenge. In contrast, hCEA-transgenic mice developed no antibody response and accepted the tumor in more than 90% of cases, thus demonstrating the role of human CEA as a foreign antigen. Accordingly, active immunization using tumor lysate or lymphocytes loaded with hCEA resulted in a CTL response and tumor-rejection in up to 80% of wild-type mice. hCEA-transgenic mice could be induced with both immunization protocols to build up a CTL response, although the number of CTL were much lower and the cytotoxic response weaker than in wild-type mice. In vivo hCEA-transgenic mice rejected hCEA-positive tumors only after immunization with the tumor lysate in about 60% whereas there was no rejection of tumors after immunization with the human hCEA-loaded autologous lymphocytes. CONCLUSION: The findings clearly show the importance of transgenic models when testing the effects of immunization towards human tumor associated antigens such as hCEA because results differ in wild-type and transgenic mice.

Animals↗
PubMed1997

Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

Chromatin↗
PubMed2003

Cortical glucose metabolism is altered in aged transgenic Tg2576 mice that demonstrate Alzheimer plaque pathology.

Alzheimer's disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of beta-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of beta-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in beta-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding beta-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparison to non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high beta-amyloid burden. This results from a reduction in glycolytic activity in beta-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.

Age Factors↗
PubMed2003

Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology.

The cleavage of APP by BACE initiates the amyloidogenic process in Alzheimer's disease (AD). We have generated transgenic mice expressing BACE and double transgenic mice expressing BACE and the Swedish mutations of APP (SwAPP) in neurons. BACE transgenic mice did not develop beta-amyloid plaques by age of 14 months, but showed intracellular beta-amyloid immunoreactivity that was co-localized with transgenic BACE in neurons. Abeta levels were increased and AD-like pathology was accelerated in double transgenic mice expressing both BACE and SwAPP. At two months of age, early signs of extracellular Abeta deposition and reactive astrocytes were found in double transgenic, but not in single transgenic mice. Furthermore, at four months, well defined beta-amyloid deposits surrounded by activated astrocytes could be detected in the double transgenic mice. We suggest that BACE overexpression is not sufficient to produce beta-amyloid plaques, but simultaneous expression of BACE and its substrate (SwAPP) leads to an accelerated amyloid plaque formation.

Amyloid Precursor Protein Secretases↗
PubMed2005

Organization, not duplication, triggers silencing in a complex transgene locus in rice.

Despite the presence in nature of many functional gene families that contain several to many highly similar sequences, the presence of identical DNA sequence repeats is widely thought to predispose transgene inserts to homology dependent gene silencing (HDGS). The induction of transcriptional gene silencing (TGS) by RNAs homologous to promoter sequences has been reported recently in Arabidopsis and humans. However, mechanisms for TGS have not been studied in detail for rice, the most widely cultivated crop plant. Taking advantage of a well-characterized homozygous silenced transgenic rice line (siJKA), supertransformation was performed with a binary vector bearing mUbi1 and 35S promoter sequences identical to those in the resident transgenes. Analysis of the incoming and resident transgenes in the supertransformants revealed that the incoming mUbi1 transgene promoter was not silenced whereas the incoming 35S transgene promoter was silenced. That the resident silenced mUbi1-bar was not reactivated in these experiments as a result of passage through tissue culture and regeneration was established by the finding that regenerants from siJKA immature embryos were all silenced for mUbi1-bar. In a parallel experiment, when wild type rice calli were transformed with the same binary vector, neither of the incoming transgene promoters was silenced. Following 5-azacytidine (5-azaC) treatment of siJKA, aberrant RNA species corresponding to the 35S promoter, but not to the mUbi1 promoter, were detected. Nevertheless, no 21-25 nt RNAs corresponding to the 35S promoter sequence were detected. These results, together with detailed analyses of the progenies from the primary transformants and supertransformants, revealed that HDGS of the resident silenced locus was caused not by simple transgene duplication, but by aberrant transcripts derived from rearranged promoters present in siJKA. Practical consequences of this study include a justification for the use of multiple copies of a given promoter for transformation without inducing silencing, provided that their genomic integration does not result in aberrant transcription of the promoters.

Azacitidine↗
PubMed1991

Transgenic mice mimic the methylation pattern of the human HLA-DR alpha gene.

The methylation pattern of the human HLA-DR alpha gene has been studied in different tissues of transgenic mice. Offspring from two transgenic lines was selected for this analysis, carrying the integrated HLA-DR alpha gene in either single or multiple (8-10) copies per diploid genome. In transgenic animals two distinct methylation patterns of the HLA-DR alpha gene are generated, due to a complete methylation of all the GCGC and CCGG sites the former, and to unmethylation restricted to one or both the GCGC sites located in the 5' portion of the HLA-DR alpha gene, the latter. Unmethylation restricted to the 5' portion of the HLA-DR alpha gene is a highly conserved feature in human tissues and in vitro cultured cell lines; therefore, it is concluded that the methylation pattern of the human HLA-DR alpha transgene may be faithfully reconstituted in transgenic animals. Northern blotting analysis of the RNA isolated from tissues of the transgenic mouse carrying single-copy HLA-DR alpha transgene demonstrates its tissue specific expression, suggesting that transgenic mice may represent an "in vivo" experimental system to study the relationship between methylation state and transcriptional activation.

Animals↗
PubMed1991

Liver-specific and position-effect expression of a retinol-binding protein-lacZ fusion gene (RBP-lacZ) in transgenic mice.

Transgenic mice carrying a liver-specific promoter fused to a nuclear-targeted lacZ reporter gene were generated. Three separate lines of mice showed liver expression in the adult and no expression elsewhere in the animal. These results show that a 1.7 kb 5'-flanking region in the retinol-binding protein gene contains necessary and sufficient transcriptional signals for expression in adult livers. A fourth line (R197) did not express the transgene in the liver; instead, constitutive lacZ expression was seen during postimplantation stages of development from Day 9.5 onwards and appeared to be associated with segmented structures including somites, branchial arches, and hindbrain rhombomeres until late fetal stages. The beta-galactosidase positive cells in R197 were later seen to give rise to facial and flank musculature, and to other region-specific subpopulations of the jaws, neocortex, and brain stem. Northern blot analysis for the host retinol-binding protein RNA transcript did not show overlapping tissue expression with the reporter gene and suggests that transgenic phenotype seen in segmented embryonic structures of R197 and other extra-hepatic sites is from novel cis-acting transcriptional specificity. RNase protection assays of the R197 mRNA indicate that the lacZ sequences are appropriately transcribed downstream of the RBP canonical TATA box, even though the RBP promoter is itself silent. This result would suggest host flanking sequences with enhancer activity may have either activated the lacZ reporter gene or cooperated with the RBP promoter to create novel region-specific transcription. Breeding experiments have so far failed to produce offsprings that are homozygous for the transgene, and mating of transgenic F1 siblings routinely produce reduced litter sizes. Embryos that are homozygous for the transgene appear to be unable to survive beyond the egg cylinder stages of development. Thus, disruption of the host genome by insertional mutation appears to be manifest at an earlier stage than when position-effect expression of the transgene is first apparent. These experiments demonstrate that the component parts of a transgene may be subject to differential activation or suppression by host genomic flanking sequences and that even a strong, tissue-specific promoter may be overridden by host genes.

Animals↗
PubMed1996

Insulin-like growth factor I expression alters acute sensitivity and tolerance to ethanol in transgenic mice.

We compared some biobehavioral effects of ethanol in transgenic mice that overexpress insulin-like growth factor I (IGF-I) in brain and in those that exhibit ectopic: brain expression of IGF binding protein I with those in non-transgenic littermate controls. Ethanol-induced sleep in IGF-I transgenic mice was significantly shorter, and in IGF binding protein 1 transgenic mice significantly longer, than in controls. A similar tendency, though not significant, was observed for ethanol-induced hypothermia. The groups did not differ in the degree of ethanol-induced ataxia. IGF-I transgenic mice did not acquire tolerance to either the hypothermic or hypnotic effects of ethanol following 7-day ethanol treatment. In contrast, tolerance in IGF binding protein 1 transgenic mice was significantly more pronounced than in controls. There were no significant differences among the three groups in the peak blood alcohol concentrations or the overall blood alcohol curves following acute ethanol challenge. In general, these data support the prediction made that chronically elevated exposure to IGF-I in IGF-I transgenic mice renders them less susceptible to the effects of ethanol than their non-transgenic siblings, and that overexpression of IGF binding protein 1 has the opposite effect.

Animals↗
PubMed1994

The use of shuttle vectors for mutation analysis in transgenic mice and rats.

The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (lambda LIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the lambda LIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity of the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of DNA damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating lambda LIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general.

Animals↗
PubMed1994

A transgenic mouse model to assess the interaction of cytotoxic T lymphocytes with virally infected, class I MHC-expressing astrocytes.

Astrocytes provide crucial support for neurons and their impairment by viruses or their interactions with anti-viral or autoimmune responses could contribute to neurological disease. We have developed a transgenic mouse model to assess lymphocyte-astrocyte interactions. The major histocompatibility complex (MHC) class I molecule, Db, was expressed in astrocytes under the transcriptional control of regulatory sequences from the glial fibrillary acidic protein (GFAP) gene. Baseline cerebral MHC class I mRNA levels from transgenic mice were elevated over those of non-transgenic controls, and a prominent increase in cerebral MHC class I expression occurred following focal, injury-induced astroglial activation within transgenic brains but not in non-transgenic controls. FACS analysis of explant astrocyte cultures from established transgenic lines demonstrated astroglial expression of the GFAP-Db fusion gene at the protein level. Functional antigen-presenting capacity was conferred by the Db transgene, as virus-infected primary astrocytes obtained from transgenic BALB/c mice (KdIdDdLd) expressing the Db molecule were lysed by Db-restricted anti-viral CTL.

Animals↗
PubMed1995

Spatial and temporal regulation of the rat calmodulin gene III directed by a 877-base promoter and 103-base leader segment in the mature and embryonal central nervous system of transgenic mice.

Three non-allelic rat calmodulin (CaM) genes CaMI, CaMII and CaMIII, which share no homology in their 5'-upstream regions, are coordinately expressed in neurons of the central nervous system (CNS). Deletion analysis of the CaMIII promoter showed that the upstream segments longer than 700 bases functioned as efficient promoters, and that the sequence from -133 to -65 was required for the activity of house-keeping type promoter in transient expression assays on a mouse glioma cell line C6. However, the transient expression seemed not to be cell type specific. To determine the temporal and spatial specificity of the promoter function, we produced transgenic mice carrying a fusion gene of the CaMIII segment from -877 to +103 and the lacZ reporter gene. In CNS of the adult transgenic mice, the localization of transgene expression was similar to that of endogenous CaMIII transcripts analyzed by in situ hybridization. The transgene was expressed prominently in pyramidal cells of the cerebral neocortex and the hippocampal regions CA1 to CA3, in Purkinje cells of the cerebellar cortex, and in neurons of the spinal cord, and moderately in granule cells of the dentate gyrus and the cerebellar cortex. In the developing CNS, the overall profiles of neuron-specific expression were also similar for both transgene and endogenous CaMIII that were expressed in the mantle layer and the dorsal root ganglia of the embryonal spinal cord. These results indicated that the neuron-specific expression of rat CaMIII was directed by this 877-base promoter sequence. The CaMIII segment used for the promoter of transgene contained a 29-bp sequence at -410, namely H3, which was conserved in the upstream regions of vertebrate CaMII and CaMIII. H3 seemed to play a pivotal role in the temporal and spatial expression of transgene in CNS, although the deletion of H3 did not decrease CAT activity in the transient expression. The transgene expression was not observed in the external granular cells of the developing cerebellum and in some neurons of the embryonic sensory ganglia in which the endogenous CaMIII was obviously expressed. Therefore, the other cis-acting element(s) located outside of this 877-bp segment seemed to be required for the temporal regulation of CaMIII in certain rudimentary neurons.

Animals↗
PubMed1993

Regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying human tyrosine hydroxylase gene.

We investigated the regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying multiple copies of human tyrosine hydroxylase (TH). The in vitro TH activity of transgenic striatum at pH 7.0 was approximately 2.8-fold higher than that of non-transgenic striatum. This augmentation is similar to that of the in vitro TH activity at pH 6.0, indicating that the expression of human TH in transgenic striatum induced little change in the phosphorylation level of TH. L-3,4-Dihydroxyphenylalanine (DOPA) formation in striatal slices of transgenic mice was approximately 2.7-fold higher than that of non-transgenic mice. The addition of 0.5 mM (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) to the incubation medium brought a negligible increase in DOPA formation in both cases. These results suggest that 6R-BH4 is not the limiting factor of TH in situ both in the transgenic and non-transgenic mice.

Animals↗
PubMed1990

The methylation-free status of a housekeeping transgene is lost at high copy number.

Transgenic mouse lines were established bearing tandem arrays of a fusion construct comprising the promoter region of a housekeeping gene, HMGCR, encoding 3-hydroxy 3-methylglutaryl CoA reductase, linked to a bacterial cat reporter gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was observed in all transgenic mouse tissues examined. The methylation state of the fusion transgene was determined. In non-transgenic mice the endogenous HMGCR promoter is devoid of methylation while flanking regions are extensively modified. In HMGCR-cat transgenic mice the fusion gene promoter was found to be similarly hypomethylated. However, the extent of hypomethylation varied with copy number: methylation-free status was progressively lost with increasing transgene copy number. Further transgenic mouse lines were constructed carrying a truncated HMGCR regulatory region linked to cat. Transgene expression and hypomethylation were observed in testis but not in any other tissue, and testis-specific methylation-free status was also lost at high copy number. Loss of hypomethylation at high copy number may indicate that saturable DNA-binding factors normally protect the HMGCR promoter from methylation.

Animals↗
PubMed1995

A 700-bp fragment of the human antithrombin III promoter is sufficient to confer high, tissue-specific expression on human apolipoprotein A-II in transgenic mice.

The human antithrombin III-encoding gene (hAT-III) promoter (phAT-III) was used to generate transgenic mice producing a human hepatic apolipoprotein, apolipoprotein A-II (hApoA-II). Integration of the transgene into the mouse genome resulted in the efficient production of hApoA-II in plasma, reaching up to 0.40 g/l in two transgenic lines. The human ApoA-II mRNA was detected at high levels, both in the liver and in the kidney of transgenic mice. The rat AT-III gene shows the same expression pattern. In contrast, as previously described, the same promoter permitted the expression of the SV40 large T antigen only in the liver of transgenic mice. In view of the extra-hepatic distribution of the ApoA-II mRNA, a preliminary characterization of the hAT-III proximal promoter (phAT-III), driving the expression of the transgene, was realized. Using DNase I footprinting analysis with liver nuclear extracts, four protected regions (I-IV) were identified in the first 175 bp of the 5' region of hAT-III. Electrophoretic mobility shift assays performed with liver and kidney nuclear extracts indicate that region III (nucleotides (nt) -67 to -90) interacts with the liver-enriched HNF4 nuclear factor. Furthermore, our data suggest that region I (nt -123 to -138) interacts with the liver-enriched HNF3 transcription factor family, both in liver and kidney. Taken together, these results demonstrate that phAT-III is a useful tool to create transgenic mice producing high plasma levels of a human apolipoprotein due to expression of the transgene in liver and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Close linkage of a transgene insertion site to the steel (Sl) locus on mouse chromosome 10.

The characterization of the insertion sites of exogenous sequences in transgenic mice can identify loci that are potentially useful for the genetic analysis of the mammalian genome. We have found that the transgene insertion site in the transgenic line TG.EB is tightly linked with the Steel (Sl) locus on mouse chromosome 10. In a backcross between doubly heterozygous transgenic Sl (Tg.EB +/+ Sl) mice and wild-type mice, only one recombinant was found in 135 progeny (recombination percentage = 0.7 +/- 0.7). The recombination frequency of the transgene with marker loci known to flank Sl was consistent with a map position close to Sl. Genomic sequences that are adjacent to the transgene insertion site were cloned and found to be tightly linked with the Sl locus in interspecific crosses using nontransgenic mice. Recombination analysis utilizing the transgene insertion site locus was used to show that a recently identified hematopoietic growth factor is encoded at Sl. The cloned sequences from the transgene insertion site are polymorphic in inbred strains of mice and can be utilized to determine the genotype at Sl during early embryonic development. Further, they may be useful in characterizing the genomic region near Sl that is affected in Sl deletion mutants.

Animals↗
PubMed1989

Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E. coli ada repair gene.

The E. coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA. After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes. Eventually, transgenic mice containing the ada fusion DNA were generated. The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross. RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses. O6MTase activity was increased in the liver of transgenic mice of line No. 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes. The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis. These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.

Animals↗