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PubMed1991

Somatic hypermutation of immunoglobulin kappa may depend on sequences 3' of C kappa and occurs on passenger transgenes.

We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism.

Amino Acid Sequence↗
PubMed1992

Tissue-specific expression and induction of human O6-alkylguanine-DNA alkyltransferase in transgenic mice.

O6-Alkylguanine-DNA alkyltransferase (ATase) activity was determined in crude sonicates of tissues obtained from the F2 offspring of human ATase transgenic founder mice. In certain cases, samples were analyzed both before and after administration of zinc sulfate in the drinking water for 2 wk to upregulate the mouse metallothionein-1 promoter that controls the expression of the transgene. In liver samples obtained by partial hepatectomy, the ATase activities of nontransgenic mice ranged from 63 to 139 fmol/mg total protein (mean of 10 mice, 95.3 +/- 23 fmol/mg), whereas in positive transgenic mice, the range was from 503 to 2119 fmol/mg (mean of 10 mice, 963 +/- 475 fmol/mg). The difference between the mean ATase values for these two groups of mice is highly significant (P less than 0.001). All positive mice expressed ATase and in those examined using the human ATase coding sequence as a probe, isoschizomeric-restriction endonuclease digestion showed no evidence of cytosine methylation in the transgene. After zinc sulfate induction, the ATase levels in residual liver tissue were for the controls 84-191 fmol/mg (mean of 10 mice, 123 +/- 31.5 fmol/mg) and for positive mice 908-3273 fmol/mg (mean of 10 mice, 1960 +/- 724 fmol/mg). Induction thus caused a 1.4- to 3.2-fold increase in ATase activity in the tissues of individual transgenic mice (mean, 1.8-fold; P less than 0.003), with the greatest increase generally occurring in those mice that had the lowest preinduction levels. Hepatic ATase levels were thus increased up to 28 times higher in transgenic mice than in nontransgenic mice. When data from other groups of transgenic and nontransgenic mice (eight of each) was included and analyzed in an independent rather than paired fashion, the mean values for zinc-treated controls and transgenic mice, respectively, were 106 fmol/mg and 1415 fmol/mg, still a highly significant (P less than 0.001) difference. In two mice given a single intraperitoneal dose of cadmium chloride, hepatic ATase increased 2.1- and 4.9-fold, respectively. The effect of partial hepatectomy alone was also considered: for transgenic mice the mean ATase level increased from 453 to 661 fmol/mg protein after 48 h. In other offspring subjected to either unilateral nephrectomy or orchidectomy, induction of ATase activity by zinc sulfate was also seen in kidney (5.7- and 8.4-fold) and testis (1.7- and 3.1-fold), although these observations were made with small numbers of mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2001

The spread of transgene expression at the site of gene construct injection.

Seven 2-day-old golden retriever pups were given focal intramuscular injections of a first generation adenovirus-dystrophin minigene construct and adenovirus-beta-galactosidase construct as a 2:1 mixture into the left anterior tibial muscle. The spread of transgene expression within the anterior tibial muscle was compared with the spread of methylene blue dye after identical injection into the contralateral muscle. Transgene expression 5-7 days after intramuscular injection was shown to extend between 5.8 and 11.6 mm along the biopsied muscle length (range of biopsy lengths 11.1-12.2 mm). The level of transgene expression at 2-2.5-mm intervals from the site of injection was significantly related to the distance from the site of injection (dystrophin, P = 0.009; beta-galactosidase, P = 0.015). The spread of methylene blue dye within the anterior tibial muscle < or =24 h after identical intramuscular injection demonstrated a similar pattern to the transgene expression, with dye staining measured between 5.5 and 8.5 mm along the muscle sample length (range of biopsy lengths 5.6-15.6 mm). The greatest transgene expression and dye staining was measured 2-2.5 mm proximal to the site of injection with a maximum of 23% of muscle fibers expressing the dystrophin transgene, 95.2% expressing the beta-galactosidase transgene, and 98% of the tissue section stained with methylene blue dye. These results suggest transgene expression after focal intramuscular injection is relatively localized around the site of injection. Further research is required to develop techniques that will provide transgene expression throughout the length and breadth of a muscle.

Adenoviridae↗
PubMed1995

Chemical skin carcinogenesis is prevented in mice by the induced expression of a TGF-beta related transgene.

Skin papillomas and squamous cell carcinomas (SCCs) are induced in mice by tumor initiation with a carcinogen followed by tumor promotion with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These usually arise from preneoplastic lesions characterized by epidermal proliferation and hyperplasia, dermal edema, and inflammation. To evaluate the role of polypeptide growth factors in chemically induced skin carcinogenesis, we used transgenic mice carrying the cDNA for a TGF-beta related molecule, bone morphogenetic protein-4 (BMP-4), under the control of the regulatory elements of the cytokeratin IV* gene in a skin carcinogenesis protocol. Control non-transgenic littermates and BMP-4 transgenic mice were treated with a single dose of a carcinogen, N-methyl-N'-nitrosoguanidine (MNNG), and biweekly with the tumor promoter TPA for 9 months. In control littermates TPA induced epidermal hyperproliferation, atypia with "dark" cells, and dermal inflammation, resulting in papillomas and SCCs in 13 of 26 animals tested. In BMP-4 transgenic mice, TPA treatment induced the expression of the BMP-4 transgene in interfollicular epidermis but only minimal epidermal thickening, hyperproliferation, and inflammation were noted after the initial dose of TPA. Furthermore, the mitotic indices in transgenic epidermis after 9 months of TPA treatment were significantly lower than the corresponding indices from untreated transgenic epidermis. Consequently, none of the 22 transgenic animals tested developed papillomas or SCCs. In conclusion, we have shown that the TPA induced expression of the BMP-4 transgene blocks proliferation and inflammation in skin, steps that are critical to the subsequent formation of papillomas and SCCs and we characterized an inducible promotersystem which expresses polypeptides in interfollicular epidermis after exogenous stimulation.

Animals↗
PubMed1997

Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.

As an economical and safer alternative to the maintenance of transgenic animals as live stocks, transgenic embryo cryobanks can be generated and maintained indefinitely. Two-cell embryos obtained from four lines of caprine beta-lactoglobulin transgenic mice homozygous for the transgene were cryopreserved, and their response to cryopreservation-related stress was investigated. Significant differences between transgenic lines were found in the viability of frozen/thawed transgenic embryos and also in two-cell embryo production after superovulatory treatment of transgenic females. The results of this study suggest that cryopreservation protocols should be assessed on each transgenic line before the generation of transgenic embryo banks.

Animals↗
PubMed1993

Expression of chimeric human transferrin-chloramphenicol acetyltransferase genes in liver and brain of transgenic mice during development.

Transferrin (TF) gene expression is tissue specific and is regulated during development. Transgenic mice have been developed which carry 1.2 or 0.67 kb of the TF 5' flanking region of the human TF gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The onset of expression of the chimeric human TF-CAT transgenes in liver and brain during development has been studied in these transgenic mice. In brain, the TF(0.67)CAT transgene began to express between 5 and 10 days after birth; in liver, the TF(0.67)CAT transgene was turned on between 10 and 20 days after birth. Endogenous mouse TF mRNA levels in liver and brain have also been measured during development by Northern analysis. In brain, the developmental expression pattern of the TF(0.67)CAT transgene is the same as the mouse endogenous TF gene; in liver, the transgene is turned on later than the endogenous mouse TF gene. DNA-protein mobility shift assays and DNase I footprinting analyses were conducted in the region of -621 to -409 bp of the human TF gene by using TF-CAT expressing liver nuclear extract from 27-day-old mice and nonexpressing liver nuclear extract from 7-day-old mice. The level of protein-DNA complex formation is several times higher in the expressing extracts, and the region from -481 to -463 bp of human TF gene is protected by the expressing extract but not the nonexpressing extracts. As demonstrated by this and other studies, the transgenic mouse model furnishes a unique opportunity to analyze developmental regulation of human transgenes.

Animals↗
PubMed2000

Radial transformation-associated recombination cloning from the mouse genome: isolation of Tg.AC transgene with flanking DNAs.

Transformation-associated recombination (TAR) cloning allows entire genes and large chromosomal regions to be specifically, accurately, and quickly isolated from total genomic DNA. We report the first example of radial TAR cloning from the mouse genome. Tg.AC mice carry a zeta-globin promoter/v-Ha-ras transgene. Fluorescence in situ hybridization localized the transgene integrant as a single site proximal to the centromere of chromosome 11. Radial TAR cloning in yeast was utilized to create orientation-specific yeast artificial chromosomes (YACs) to explore the possibility that cis-flanking regions were involved in transgene expression. YACs containing variable lengths of 5' or 3' flanking chromosome 11 DNA and the Tg.AC transgene were specifically chosen, converted to bacterial artificial chromosomes (BACs), and assayed for their ability to promote transcription of the transgene following transfection into an FVB/N carcinoma cell line. A transgene-specific reverse transcription-polymerase chain reaction assay was utilized to examine RNA transcripts from stably transfected clones. All Tg.AC BACs expressed the transgene in this in vitro system. This report describes the cloning of the v-Ha-ras transgene and suggests that transcriptional activity may not require cis elements flanking the transgene's integration site.

Animals↗
PubMed1998

Transgenic A1 adenosine receptor overexpression markedly improves myocardial energy state during ischemia-reperfusion.

A1 adenosine (A1AR) activation may reduce ischemia-reperfusion injury. Metabolic and functional responses to 30 min global normothermic ischemia and 20 min reperfusion were compared in wild-type and transgenic mouse hearts with approximately 100-fold overexpression of coupled cardiac A1ARs. 31P-NMR spectroscopy revealed that ATP was better preserved in transgenic v wild-type hearts: 53 +/- 11% of preischemic ATP remained after ischemia in transgenic hearts v only 4 +/- 4% in wild-type hearts. However, recovery of ATP after reperfusion was similar in transgenic (46 +/- 5%) and wild-type hearts (37 +/- 12%). Reductions in phosphocreatine (PCr) and cytosolic pH during ischemia were similar in both groups. However, recovery of PCR on reperfusion was higher in transgenic (67 +/- 8%) v wild-type hearts (36 +/- 8%), and recovery of pH was greater in transgenic (pH = 7.11 +/- 0.05) v wild-type hearts (pH = 6.90 +/- 0.02). Bioenergetic state ([ATP]/[ADP].[Pi]) was higher in transgenic v wild-type hearts during ischemia-reperfusion. Time to ischemic contracture was prolonged in transgenic (13.6 +/- 0.8 min) v wild-type hearts (10.4 +/- 0.3 min). Degree of contracture was lower and recovery of function in reperfusion higher in transgenic v wild-type hearts. In conclusion, A1AR overexpression reduces ATP loss and improves bioenergetic state during severe ischemic insult and reperfusion. These changes may contribute to improved functional tolerance.

Animals↗
PubMed1999

Plasmacytoma induction in specific pathogen-free (SPF) bcl-2 transgenic BALB/c mice.

Germ-free (GF) and specific pathogen free (SPF) BALB/c mice are refractory to plasmacytoma induction by pristane (McIntire and Princler, 1969, Byrd et al 1991). It was therefore suggested that MPC development may depend on antigenic stimulation. If so, it may conceivably act by preventing the apoptotic elimination of tumor precursor cells. We have tested this idea by elevating the apoptotic threshold by the introduction of a bcl-2 transgene. We have found that MPCs could be induced by pristane oil in transgene carrying SPF mice. An E mu activated bcl-2 transgene was introduced into SPF BALB/c mice. The mice were used after two backcrosses (BC-2). Pristane oil treatment was started at 4 to 6 weeks of age (3 x 0.3 ml via i.p. at monthly intervals). For each transgene carrier a transgene negative littermate was used as control. Fifteen of 24 (63%) transgene carriers developed plasmacytomas after latency periods between 67 and 146 days (mean = 112 +/- 30 days) after the first pristane injection. Five additional transgene carriers developed lymphoma (3 cases) or mixed MPC and lymphoma (2 cases). In contrast, no tumors developed in 16 transgene negative littermates that were kept > 300 days under observation. Karyotyping showed that 10/15 (66%) of the MPCs carried a T(12;15) translocation, 4/15 (27%) carried both T(12;15) and T(6;15) translocations in the same metaphase plate, and 1/15 (7%) was translocation free. A T(12;15) translocation was also detected in one of the 2 mice with mixed tumor type. Pristane treated bcl-2 transgenic C57B1/6 mice remained tumor free, although T(12;15) translocation carrying cells were found in the peritoneal fluid of 4/20 mice 176 days after pristane.

Animals↗
PubMed1995

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Animals↗
PubMed1994

Ectopic expression of beta-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation and in situ localization.

We produced transgenic mice carrying the native sheep beta-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains. In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

Animals↗
PubMed1992

Expression of human serum albumin in the milk of transgenic mice.

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep beta-lactoglobulin (BLG) 5'-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5'-sequences demonstrated that 3 kb of 5'-sequences were sufficient to support high levels of expression of BLG, and that the longer 5'-sequences did not improve upon the levels of expression. As such, the 3 kb 5'-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml-1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 micrograms ml-1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.

Animals↗
PubMed1996

Transgenic mice expressing human phospholipid transfer protein have increased HDL/non-HDL cholesterol ratio.

The role of plasma phospholipid transfer protein (PLTP) in lipoprotein metabolism is poorly understood. In vitro studies suggest that PLTP influences HDL size and composition and transfers phospholipids among lipoproteins. To provide an in vivo model for studies of PLTP physiology, transgenic mice that express human PLTP were generated. Human PLTP transcripts were detected in total RNA from adipose tissue, lung, heart, and spleen of the two distinct lines (A and C) of transgenic mice. Despite minimal expression of human PLTP in the liver of these transgenic mice and similar plasma phospholipid transfer activity in transgenic and non-transgenic mice (19.1 +/- 3.1 vs 18.9 +/- 2.7 mumol/ml/h), differences in lipoprotein levels were observed between transgenic and control mice receiving the same chow diet. Male transgenic mice of line C had significantly higher HDL cholesterol than control mice (76.4 +/- 4.6 vs 71.9 +/- 7.0 mg/dl, p < 0.05) and the male transgenic mice of lines A and C had a significantly lower non-HDL cholesterol (15.1 +/- 4.1 and 15.6 +/- 4.7 vs 20.9 +/- 5.5 mg/dl, P < 0.01 and P < 0.02) and a significantly higher HDL cholesterol/non-HDL cholesterol ratio than the control mice (5.3 +/- 1.3 and 5.5 +/- 2.2 vs 3.9 +/- 1.9 mg/dl, P < 0.01 and P < 0.02). Female mice from transgenic line C had higher HDL cholesterol than control mice (64.6 +/- 4.8 vs 57.4 +/- 5.1 mg/dl, P < 0.01) while female mice from line A tended to have higher HDL cholesterol/non-HDL cholesterol ratio than control mice (5.5 +/- 3.7 vs 3.8 +/- 1.4). These observations suggest that expression of PLTP in peripheral tissues play an important role in lipoprotein metabolism. Expression of human PLTP produced a more favorable lipoprotein profile and thus, enhanced expression of PLTP could potentially retard atherosclerosis.

Animals↗
PubMed2003

Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup.

Transgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance ( bar) or antibiotic resistance ( aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes ( gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15-20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T(0) plants or T(1) progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.

Agriculture↗
PubMed2006

Development of allele-specific PCR and RT-PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model.

Members of the NBS-LRR gene family impart resistance to a wide variety of pathogens and are often found clustered within a plant genome. This clustering of homologous sequences can complicate PCR-based characterizations, especially the study of transgenes. We have developed allele-specific PCR and RT-PCR assays for the potato late blight resistance gene RB. Our assay utilizes two approaches toward primer design, allowing discrimination between the RB transgene and both the endogenous RB gene and numerous RB homeologs. First, a reverse primer was designed to take advantage of an indel present in the RB transgene but absent in rb susceptibility alleles, enhancing specificity for the transgene, though not fully discriminating against RB homeologs. Second, a forward primer was designed according to the principles of mismatch amplification mutation assay (MAMA) PCR, targeting SNPs introduced during the cloning of RB. Together, the indel reverse primer and the MAMA forward primer provide an assay that is highly specific for the RB transgene, being capable of distinguishing the transgene from all RB endogenous gene copies and from all RB paralogs in a diverse collection of wild and cultivated potato genotypes. These primers have been successfully multiplexed with primers of an internal control. The multiplexed assay is useful for both PCR and RT-PCR applications. Double MAMA-PCR, in which both PCR primers target separate transgene-specific SNPs, was also tested and shown to be equally specific for the RB transgene. We propose extending the use of MAMA for the characterization of resistance transgenes.

Alleles↗
PubMed2000

Transgenic mice overexpressing alpha2A-adrenoceptors in pancreatic beta-cells show altered regulation of glucose homeostasis.

AIMS/HYPOTHESIS: To study the role of the human alpha2A-adrenoceptor in the regulation of insulin secretion and the maintenance of glucose homeostasis in transgenic mice overexpressing this receptor in pancreatic beta cells. METHODS: A human insulin promoter/human alpha2C10-adrenoceptor chimeric gene was microinjected into mouse embryos and transgenic mice were obtained. RESULTS: Analysis by RT-PCR showed that the expression of the transgene was restricted to pancreatic islets. Study of the binding of the alpha2-antagonist [3H]RX821002 to membrane preparations showed that islets from transgenic mice had ninefold higher alpha2-adrenoceptor density than those from controls. Immunohistological analysis showed, however, no change in the number or size of islets between control and transgenic mice. Transgenic animals had normal glycaemia and insulinaemia in basal conditions but greater hyperglycaemic and hypoinsulinaemic responses after injection of the alpha2-agonist, UK14304. The lower blood insulin concentration detected in transgenic mice was a reflection of a stronger inhibitory effect of the alpha2-agonist on glucose-stimulated insulin secretion in transgenic islets than in controls. Furthermore, transgenic mice did not have lower glycaemia to basal values after an intraperitoneal glucose tolerance test. This defect was abolished by treatment with the alpha2-adrenoceptor antagonist, RX821002. CONCLUSION/INTERPRETATION: These results provide evidence in vivo that overexpression of alpha2-adrenoceptors in beta cells can lead to impaired insulin secretion and glucose intolerance.

Adrenergic alpha-Agonists↗
PubMed2004

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques.

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

Agriculture↗
PubMed1999

A method of tracking donor cells after simulated autologous transplantation: a study using synovial cells of transgenic rats.

A transgenic rat was used as a transplantation donor to simulate autologous transplantation. The sex-matched transplantation between a female transgenic and a wild-type rat can theoretically be regarded as an autologous transplantation due to the genetic agreement of these rats except for the non-protein-producing transgenes. Transgene-containing synovial cells were tracked in the joint using this autologous transplantation model. The transgenes in the donor synovial cells were detected using in situ hybridization (ISH), while mitotic activities were simultaneously examined by immunodetection of 5-bromo-2'-deoxyuridine (BrdU). A defect was generated in the knee joint capsule of a Fischer 344 (wild-type) rat. The synovium of a transgenic rat was sutured to the defect of the wild-type rat in group 1 and was allowed to free float in the joint in group 2. A large number of BrdU-labeled, transgene-containing synovial cells were detected in both groups at 3 days. The number of these cells then decreased, but they could still be identified even at 4 weeks after autologous transplantation. These results indicated that transplanted synovial cells were viable in the joint for at least 4 weeks. Furthermore, the transgenic rat was shown to be an effective animal model for distinguishing the extrinsic from the intrinsic cells in the cellular intermixed tissues in vivo. The combined method of ISH for detecting transgene-containing cells and the immunohistochemistry of BrdU for detecting proliferating cells was also shown to be effective for tracking the viability of extrinsic cells after autologous transplantation.

Animals↗