Hemizygous Transgenic Mice Research Articles

Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonatal mice from a lethal challenge with enterovirus type 71

The human Enterovirus genus of the piconavirus family causes most of the febrile illnesses that affect children during the summer season in Taiwan. Enterovirus type 71 (EV71) plays a key role in patients with hand-foot-and-mouth disease (HFMD) combined with severe paralysis or encephalitis. It is important to find a method for preventing infection with EV71 since there is no antiviral agent or vaccine for humans. In this study, we developed a transgenic mouse model for demonstrating the protective effects of recombinant lactoferrin (LF) against EV71 infection. Transgenic mice carrying alpha-lactalbumin-porcine lactoferrin (alphaLA-pLF) and BALB/c wild-type mice were subjected to EV71 inoculation. First, we analyzed the expression efficiencies of recombinant pLF (rpLF) in hemizygous and homozygous transgenic mice. Following EV71 inoculation on the 4th day of life, pups ingesting transgenic milk showed the significantly higher survival rate and heavier body weight compared to wild-type mice. RT-PCR analysis for EV71 viral RNA showed that the recombinant pLF had a blocking effect on EV71 infection. Our data suggest that oral intake of pLF-enriched milk exhibited the ability to prevent infection with EV71. The study also provides an animal model for validating the protective effects of pLF.

Cell lines from MYCN transgenic murine tumours reflect the molecular and biological characteristics of human neuroblastoma

Overexpression of the human MYCN oncogene driven by a tyrosine hydroxylase promoter causes tumours in transgenic mice that recapitulate the childhood cancer neuroblastoma. To establish an in vitro model to study this process, a series of isogenic cell lines were developed from these MYCN-driven murine tumours. Lines were established from tumours arising in homozygous and hemizygous MYCN transgenic mice. Hemizygous tumours gave rise to cell lines growing only in suspension. Homozygous tumours gave rise to similar suspension lines as well as morphologically distinct substrate-adherent lines characteristic of human S-type neuroblastoma cells. FISH analysis demonstrated selective MYCN transgene amplification in cell lines derived from hemizygous mice. Comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) analysis confirmed a range of neuroblastoma-associated genetic changes in the various lines, in particular, gain of regions syntenic with human 17q. These isogenic lines together with the transgenic mice thus represent valuable models for investigating the biological characteristics of aggressive neuroblastoma.

Cthrc1 Is a Novel Inhibitor of Transforming Growth Factor-β Signaling and Neointimal Lesion Formation

We identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene expressed in the adventitia and neointima on arterial injury and found that it functionally increases cell migration while reducing collagen deposition. To address the in vivo role of Cthrc1, we generated transgenic mouse lines that constitutively overexpress Cthrc1. An intercross of 2 transgenic lines produced offspring with brittle bones caused by a reduction in collagenous bone matrix. Hemizygous Cthrc1 transgenic mice developed normally but neointimal lesion formation and adventitial collagen deposition in response to carotid artery ligation were significantly reduced compared with wild-type littermates. In 75% of Cthrc1 transgenic mice, cartilaginous metaplasia of medial smooth muscle cells was observed as assessed by Alcian blue staining and expression of the chondrocyte marker collagen type II. Transforming growth factor-beta signaling was reduced in smooth muscle cells of Cthrc1 transgenic arteries, as demonstrated by reduced phospho-Smad2/3 immunoreactivity, whereas Smad signaling related to bone morphogenetic proteins was unaffected. Similarly, primary smooth muscle cells and PAC1 smooth muscle cells overexpressing Cthrc1 had reduced levels of phospho-Smad2/3 as well as procollagen. Furthermore, Cthrc1 inhibited transforming growth factor-beta-sensitive reporter constructs in smooth muscle but not endothelial cells. These data indicate that Cthrc1 is a cell-type-specific inhibitor of transforming growth factor-beta, which in turn impacts collagen type I and III deposition, neointimal formation, and dedifferentiation of smooth muscle cells.

Simultaneous Dystrophin and Dysferlin Deficiencies Associated with High-Level Expression of the Coxsackie and Adenovirus Receptor in Transgenic Mice

The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, is usually confined to the sarcolemma at the neuromuscular junction in mature skeletal muscle fibers. Previously, we reported that adenovirus-mediated gene transfer is greatly facilitated in hemizygous transgenic mice with extrasynaptic CAR expression driven by a muscle-specific promoter. However, in the present study, when these mice were bred to homozygosity, they developed a severe myopathic phenotype and died prematurely. Large numbers of necrotic and regenerating fibers were present in the skeletal muscle of the homozygous CAR transgenics. The myopathy was further characterized by increased levels of caveolin-3 and beta-dystroglycan and decreased levels of dystrophin, dysferlin, and neuronal nitric-oxide synthase. Even the hemizygotes manifested a subtle phenotype, displaying deficits in isometric force generation and perturbed mitogen-activated protein kinase (MAPK-erk1/2) activation during contraction. There are few naturally occurring or engineered mouse lines showing as severe a skeletal myopathy as observed with ectopic expression of CAR in the homozygotes. Taken together, these findings suggest that substantial overexpression of CAR may lead to physiological dysfunction by disturbing sarcolemmal integrity (through dystrophin deficiency), impairing sarcolemmal repair (through dysferlin deficiency), and interfering with normal signaling (through alterations in caveolin-3 and neuronal nitric-oxide synthase levels).

Transgenic expression of B‐APP in fast‐twitch skeletal muscle leads to calcium dyshomeostasis and IBM‐like pathology

Intracellular deposition of the beta-amyloid (Abeta) peptide is an increasingly recognized pathological hallmark associated with neurodegeneration and muscle wasting in Alzheimer's disease (AD) and inclusion body myositis (IBM), respectively. Previous reports have implicated dysregulation of beta-amyloid precursor protein (betaAPP) expression in IBM. Accumulation of full-length betaAPP, its various proteolytic derivatives including Abeta, and phospho-tau into vacuolated inclusions is an early pathogenic event. We previously reported on a statistical tendency favoring fast twitch fiber involvement in IBM, reminiscent of the tissue specific patterns of misfolded protein deposition seen in neurodegenerative diseases. To test this principle, we generated an animal model in which human wild-type (WT) betaAPP expression was limited to postnatal type II skeletal muscle. Hemizygous transgenic mice harboring increased levels of holo betaAPP751 and Abeta in skeletal muscle fibers became significantly weaker with age compared with nontransgenic littermates and exhibited typical myopathic features. A subpopulation of dissociated muscle fibers from transgenic mice exhibited a 2-fold increase in resting calcium and membrane depolarization compared with nontransgenic littermates. Taken together, these data indicate that overexpression of human betaAPP in fast twitch skeletal muscle of transgenic mice is sufficient for the development of some features characteristic of IBM, including abnormal tau histochemistry. The increase in resting calcium and depolarization are novel findings, suggesting both a mechanism for the weakness and an avenue for therapeutic intervention in IBM.

A 191-kb genomic fragment containing the human α-globin locus can rescue α-thalassemic mice

A 191-kb human bacterial artificial chromosome (BAC) containing the human alpha-globin genomic locus was used to generate transgenic mice that express, exclusively, human alpha-globin ((hu)alpha-globin). Expression of (hu)alpha-globin reaches a level of 36% of that of endogenous mouse alpha-globin ((mu)alpha-globin) on a heterozygous mouse alpha-thalassemia background ((mu)alpha-globin knockout, (mu)alpha(+/-)). Hemizygous transgenic mice carrying the (hu)alpha-globin locus on a heterozygous knockout background ((hu)alpha(+/0), (mu)alpha(++/--)) demonstrated complementation of most hematologic parameters. By crossing (hu)alpha(+/0), (mu)alpha(++/--) mice, we were able to generate mice entirely dependent on (hu)alpha-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous (mu)alpha-globin knockout embryos ((mu)alpha(--/--)). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the (hu)alpha-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of (hu)alpha-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating (hu)alpha-globin production and for development of therapeutic strategies for beta-thalassemia based on downregulation of alpha-globin expression.

Coincident thresholds of mutant protein for paralytic disease and protein aggregation caused by restrictively expressed superoxide dismutase cDNA

Familial amyotrophic lateral sclerosis (FALS) has been modeled in transgenic mice by introducing mutated versions of human genomic DNA encompassing the entire gene for Cu,Zn superoxide dismutase (SOD1). In this setting, the transgene is expressed throughout the body and results in mice that faithfully recapitulate many pathological and behavioral aspects of FALS. By contrast, transgenic mice made by introducing recombinant vectors, encoding cDNA genes, that target mutant SOD1 expression to motor neurons, only, or astrocytes, only, do not develop disease. Here, we report that mice transgenic for human SOD1 cDNA with the G37R mutation, driven by the mouse prion promoter, develop motor neuron disease. In this model, expression of the transgene is highest in CNS (both neurons and astrocytes) and muscle. The gene was not expressed in cells of the macrophage lineage. Although the highest expressing hemizygous transgenic mice fail to develop disease by 20 months of age, mice homozygous for the transgene show typical ALS-like phenotypes as early as 7 months of age. Spinal cords and brain stems from homozygous animals with motor neuron disease were found to contain aggregated species of mutant SOD1. The establishment of this SOD1-G37R cDNA transgenic model indicates that expression of mutant SOD1 proteins in the neuromuscular unit is sufficient to cause motor neuron disease. The expression levels required to induce disease coincide with the levels required to induce the formation of SOD1 aggregates.

Triglyceride‐lowering Effect of Respiratory Uncoupling in White Adipose Tissue

Hypolipidemic drugs such as bezafibrate and thiazolidinediones are known to induce the expression of mitochondrial uncoupling proteins (UCPs) in white adipose tissue. To analyze the potential triglyceride (TG)-lowering effect of respiratory uncoupling in white fat, we evaluated systemic lipid metabolism in aP2-Ucp1 transgenic mice with ectopic expression of UCP1 in adipose tissue. Hemizygous and homozygous transgenic mice and their nontransgenic littermates were fed chow or a high-fat diet for up to 3 months. Total TGs, nonesterified fatty acids, and the composition of plasma lipoproteins were analyzed. Hepatic TG production was measured in mice injected with Triton WR1339. Uptake and the use of fatty acids were estimated by measuring adipose tissue lipoprotein lipase activity and fatty acid oxidation, respectively. Adipose tissue gene expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. Transgene dosage and the high-fat diet interacted to markedly reduce plasma TGs. This was reflected by decreased concentrations of very-low-density lipoprotein particles in the transgenic mice. Despite normal hepatic TG secretion, the activity of lipoprotein lipase in epididymal fat was enhanced by the high-fat diet in the transgenic mice in a setting of decreased re-esterification and increased in situ fatty acid oxidation. Respiratory uncoupling in white fat may lower plasma lipids by enhancing their in situ clearance and catabolism.

332 ENHANCEMENT OF GROWTH PERFORMANCE IN DOUBLE TRANSGENIC MICE WITH GROWTH HORMONE RECEPTOR AND IGF-1 RECEPTOR GENES

The effect of amplifying growth-related receptor signaling, through overexpression of receptors, on growth regulation in animals was examined. Transgenic mice lines were produced by DNA microinjection using the metallothionein promoter ligated to either the growth hormone receptor (GHR) or IGF-1 receptor (IGF-1R) genes (3 GHR founders and 3 IGF-1R founders). Transgenic mouse lines were estimated to contain approximately 4 to 20 copies of transgenes per cell by Southern blot analysis. Founder mice of each transgenic line transmitted transgenes into F1 and F2 pups with Mendelian ratio. Double transgenic (IGF-1R/GHR) mice were produced by the mating between nine pairs of IGF-1R and GHR hemizygous transgenic F1 mice. The transmission patterns in the 78 F2 pups produced from these matings were 20 with no transgene (25.6%), 17 with the IGF-1R gene (21.8%), 25 with the GHR gene (32.1%), and 16 with both GHR and IGF-1R genes (20.5%). The mRNA expression of transgenes using RT-PCR with the specific primers for IGF-IR and GHR genes was checked in tissues of transgenic mice. Double transgenic mice with IGF-IR and GHR genes expressed more mRNAs of transgenes than non-transgenic or single transgenic mice. Growth of double transgenic mice was fastest compared with single transgenic mice containing IGF-1R or GHR genes. And GHR transgenic mice grew faster than IGF-1R transgenic mice. When body weights of 15 transgenic mice for each transgenic line were measured at 4, 10, and 14 weeks after birth, double transgenic mice were significantly heavier compared with non-transgenic control mice at each stage (24 to 30% heavier in double transgenic mice; 15 to 20% heavier in single transgenic mice, P < 0.05). These results suggest that overexpression of growth-related receptor genes could promote the growth of transgenic animals with an additive effect.

Loss of Redox Factor 1 Decreases NF-κB Activity and Increases Susceptibility of Endothelial Cells to Apoptosis

The aim of this project was to test the hypothesis that redox factor 1 (Ref-1) was a critical upstream determinant of NF-kappaB-dependent survival signaling pathways in the vessel wall. Aortas from hemizygous transgenic mice harboring a single allele of Ref-1 exhibited a significant loss in NF-kappaB DNA binding activity. The NF-kappaB-dependent survival gene A20 was significantly downregulated in aortas of hemizygous Ref-1 mice, whereas IAP-2 was unchanged. Overexpression of A20 rescued cells from tumor necrosis factor (TNF)-induced apoptosis, suggesting that the loss of A20 in Ref-1 hemizygotes may be a rate-determining step in endothelial cell fate. Deletion of the previously defined redox-sensitive or the AP endonuclease domains of Ref-1 significantly decreased NF-kappaB transcriptional activation and endothelial cell survival. Furthermore, TNF-induced apoptosis was significantly potentiated in endothelial cells after delivery of Morpholino antisense oligodeoxynucleotides targeted to Ref-1. Loss of the redox-sensitive domain blocked the ability of Ref-1 to reduce p50; however, loss of the endonuclease domain did not effect p50 reduction, suggesting alternative mechanisms of action of Ref-1 on NF-kappaB activity. These findings establish a role for Ref-1 as an upstream determinant of NF-kappaB and A20-dependent signaling and endothelial survival in the vessel wall.

Characterization of hemizygous SOD1/wild-type transgenic mice with the SHIRPA primary screen and tests of sensorimotor function and anxiety

SOD1 is one of several overexpressed genes in Down's syndrome. In order to dissect genetic causes of the syndrome, hemizygous human wild-type SOD1 transgenic mice were compared to FVB/N non-transgenic controls at 3 months of age in the SHIRPA primary screen of neurologic function as well as in tests of motor activity and coordination. The responsiveness of SOD1/wt transgenic mice to visual and somatosensory stimuli was reduced in placing, pinna, corneal, and toe-pinch tests. In addition, SOD1/wt transgenic mice crossed fewer segments on a stationary beam. On the contrary, there was no intergroup difference for motor activity and anxiety in open-field and emergence tests and for latencies before falling on the stationary beam, coat-hanger, and rotorod. These results indicate mild deficits in sensorimotor responsiveness in a mouse model expressing human SOD1 and that the overexpressed gene may be responsible for some Down symptoms.

Knock-down of the 37-kDa/67-kDa laminin receptor in mouse brain by transgenic expression of specific antisense LRP RNA.

The 37-kDa/67-kDa laminin receptor (LRP/LR) plays a major role in the propagation of PrPSc, the abnormal form of the prion protein. In order to ablate the expression of LRP/LR in mouse brain we generated transgenic mice ectopically expressing antisense LRP RNA in the brain under control of the neuron-specific enolase (NSE) promoter. Hemizygous transgenic mice TgN(NSEasLRP)2 showed a significant reduction of LRP/LR protein levels in hippocampal and cerebellar brain regions. These mice might act as powerful tools to investigate the role of the laminin receptor in scrapie pathogenesis.

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1

BackgroundCu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in the metabolism of reactive oxygen species (ROS) and pathogenetically relevant for several disease states including Down syndrome (DS; trisomy 21). Systematically studying protein expression in human brain and animal models of DS we decided to carry out "protein hunting" for hypothetical proteins, i.e. proteins that have been predicted based upon nucleic sequences only, in a transgenic mouse model overexpressing human SOD1.ResultsWe applied a proteomics approach using two-dimensional electrophoresis (2-DE) with in-gel digestion of spots followed by mass spectrometric (matrix-assisted laser desorption/ionization-time of flight) identification and quantification of hypothetical proteins using specific software. Hippocampi of wild type, hemizygous and homozygous SOD1 transgenic mice (SOD1-TGs) were analysed.We identified fourteen hypothetical proteins in mouse hippocampus. Of these, expression levels of 2610008O03Rik protein (Q9D0K2) and 4632432E04Rik protein (Q9D358) were significantly decreased (P < 0.05 and 0.001) and hypothetical protein (Q99KP6) was significantly increased (P < 0.05) in hippocampus of SOD1-TGs as compared with non-transgenic mice.ConclusionsThe biological meaning of aberrant expression of these proteins may be impairment of metabolism, signaling and transcription machinery in SOD1-TGs brain that in turn may help to explain deterioration of these systems in DS brain.

No superoxide dismutase activity of cellular prion protein in vivo.

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C), which is encoded by the Prnp gene. PrP(C) is highly expressed on neurons and its function is unknown. Recombinant PrP(C) was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrP(C), and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrP(C). We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosome-enriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrPc with mice overexpressing human Cu2+/Zn2+-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrP(C) in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.

Combined Antiretroviral Therapy Causes Cardiomyopathy and Elevates Plasma Lactate in Transgenic AIDS Mice

Highly active antiretroviral therapy (HAART) is implicated in cardiomyopathy (CM) and in elevated plasma lactate (LA) in AIDS through mechanisms of mitochondrial dysfunction. To determine mitochondrial events from HAART in vivo, 8-week-old hemizygous transgenic AIDS mice (NL4–3Δ gag/pol; TG) and wild-type FVB/n littermates were treated with the HAART combination of zidovudine, lamivudine, and indinavir or vehicle control for 10 days or 35 days. At termination of the experiments, mice underwent echocardiography, quantitation of abundance of molecular markers of CM (ventricular mRNA encoding atrial natriuretic factor [ANF] and sarcoplasmic calcium ATPase [SERCA2]), and determination of plasma LA. Myocardial histologic features were analyzed semiquantitatively and results were confirmed by transmission electron microscopy. After 35 days in the TG + HAART cohort, left ventricular mass increased 160% by echocardiography. Molecularly, ANF mRNA increased 250% and SERCA2 mRNA decreased 57%. Biochemically, LA was elevated (8.5 ± 2.0 mm). Pathologically, granular cytoplasmic changes were found in cardiac myocytes, indicating enlarged, damaged mitochondria. Findings were confirmed ultrastructurally. No changes were found in other cohorts. After 10 days, only ANF was elevated, and only in the TG + HAART cohort. Results show that cumulative HAART caused mitochondrial CM with elevated LA in AIDS transgenic mice.

Overexpression of Cu2+/Zn2+ superoxide dismutase protects against early diabetic glomerular injury in transgenic mice.

Ex vivo and in vitro observations implicate superoxide as a mediator of cell injury in diabetes, but in vivo evidence is lacking. In the current studies, parameters of glomerular injury were examined in hemizygous nondiabetic transgenic mice (SOD) and streptozotocin-diabetic (D) transgenic mice (D-SOD), which overexpress human cytoplasmic Cu2+/Zn2+ superoxide dismutase (SOD-1), and in corresponding wild-type littermates (WT, D-WT) after 4 months of diabetes. In both SOD and D-SOD mice, renal cortical SOD-1 activity was twofold higher than values in the WT mice; blood glucose and glycosylated hemoglobin (GHb) levels did not differ in the two diabetic groups. Urinary albumin excretion, fractional albumin clearance, urinary transforming growth factor-beta (TGF-beta) excretion, glomerular volume, glomerular content of immunoreactive TGF-beta, and collagen alpha1 (IV) and renal cortical malondialdehyde (MDA) levels were significantly higher in D-WT mice compared with corresponding values in D-SOD mice. Glomerular volume, glomerular content of TGF-beta and collagen IV, renal cortical MDA, and urinary excretion of TGF-beta in D-SOD mice did not differ significantly from corresponding values in either the nondiabetic SOD or WT mice. In separate groups of mice studied after 8 months of diabetes, mesangial matrix area, calculated as a fraction of total glomerular tuft area, and plasma creatinine were significantly higher in D-WT but not in D-SOD mice, compared with corresponding values in the nondiabetic mice. In vitro infection of mesangial cells (MC) with a recombinant adenovirus encoding human SOD-1 increased SOD-1 activity threefold over control cells and prevented the reduction of aconitase activity, an index of cellular superoxide, and the increase in collagen synthesis that otherwise occurred in control MC in response to culture with 300 or 500 mg/dl glucose. Thus, increases in cellular SOD-1 activity attenuate diabetic renal injury in vivo and also prevent stimulation of MC matrix protein synthesis induced in vitro by high glucose.

Growth inhibition in giant growth hormone transgenic mice by overexpression of insulin-like growth factor-binding protein-2.

To clarify the role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in postnatal growth regulation, we crossed hemizygous CMV-IGFBP-2 transgenic mice with hemizygous PEPCK-bGH transgenic mice, which are characterized by serum GH levels in the range of 2 microgram/ml. Four genetic groups were obtained: animals carrying both transgenes (GB), the GH (G) or the IGFBP-2 transgene (B), and nontransgenic controls (C). Male offspring were analyzed for serum levels of IGF-I, for serum and tissue levels of IGFBP-2, and for body and organ growth. Serum IGF-I levels were 2- to 3-fold increased (P < 0.001) in the GH-overexpressing groups, with no difference between G and GB mice. Serum IGFBP-2 levels were 4- to 9-fold (P < 0.001) increased both in B and GB vs. C and G mice. Western immunoblot analysis did not reveal differences in tissue IGFBP-2 levels between B and GB mice. IGFBP-2 levels were highest in pancreas, followed by skeletal muscle, heart, kidney, brain, skin, and spleen. No elevation of IGFBP-2 was found in the liver. Body weight gain of G and GB mice was significantly increased vs. C and B mice, resulting in almost 2-fold increased body weights at the age of 15 weeks. However, there was a significant reduction in body weight of GB vs. G mice (17%; P < 0.001) and of B vs. C mice (13%; P < 0.05). This was primarily caused by a marked reduction of carcass weight (GB vs. G, 27%; B vs. C, 21%; P < 0.001). Measurements of nose-rump-length, organ (brain, heart, spleen, liver, pancreas, kidney), and tissue weights (skin, carcass, abdominal fat) in 5- and 15-week-old mice revealed several indications that the growth-inhibiting effect of IGFBP-2 overexpression was more marked in high-GH/IGF-I mice: 1) At 5 weeks of age, GB mice displayed a significant reduction of all growth parameters except for the weight of abdominal fat, when compared with G mice, whereas only brain weight was significantly reduced in B vs. C mice. 2) In 15-week-old animals, a significant reduction in all growth parameters, except for spleen and abdominal fat weights, was seen in GB vs. G mice, whereas only nose-rump-length and the weights of carcass and brain were significantly reduced in B vs. C mice. Our study demonstrates, for the first time, the potential of IGFBP-2 to inhibit GH-stimulated growth in giant transgenic mice, providing further evidence for an inhibitory effect of this IGFBP in vivo.

Segregation ratios and growth rate in inactive ovine metallothionein 1a-ovine growth hormone transgenic mice.

Objectives were to determine whether the oMt1a-oGH transgene shows normal Mendelian segregation and whether oMt1a-oGH mice exhibit normal growth without the zinc supplementation required to increase plasma oGH levels and stimulate growth. Transgenic mice were reciprocally backcrossed for four generations to high growth and control lines to form lines GM and GR, respectively. In the fifth generation, hemizygous transgenic mice (T/-) were crossed within each line. Pooled across backcross generations, there was a deficit (P < 0.001) of T/- progeny in lines GM (31.6%) and GR (22.2%) compared with expected (50%). In the T/- x T/- cross, the combined percentage of homozygous (T/T) and hemizygous transgenic mice was less (P < 0.001) than expected (75%) in both GM (44.2%) and GR (38.5%). Backcross T/- mice had lower (P < 0.05) 3-wk BW and lower (P < 0.001) 6-wk BW and 3- to 6-wk postweaning gains than nontransgenic mice. Similar genotypic differences were found in the T/- x T/- cross. No significant growth differences were found between T/T and T/- progeny. Using segregation ratios from the T/- x T/- mating, the relative fitness estimates of T/T, T/-, and -/- (nontransgenic) mice were 0.345, 0.223, and 1.0, respectively, in line GM and 0.218, 0.205, and 1.0 in line GR. Fitness estimates in the back-cross for T/- and -/- were 0.463 and 1.0 in line GM and 0.285 and 1.0 in line GR. Abnormal segregation ratios may be due to germline mosaicism or reduced fitness due to differential embryo survival. Reduced growth of oMt1a-oGH transgenic mice when the transgene is switched off suggests a subtle developmental abnormality, which may contribute to a reduction in fitness.

The rasH2 transgenic mouse: nature of the model and mechanistic studies on tumorigenesis.

The rasH2 mouse is a hemizygous transgenic mouse carrying the c-Ha-ras oncogene and that gene's promotor/enhancer within the genetic background of a BALB/cByJ x C57BL/6J F1 mouse. Approximately 3 copies of the transgene are integrated in a tandem array into chromosome number 15. The transgene is transmitted stably without point mutation in hot spots and is expressed in all tissues over 20 backcross generations. The homozygous c-Ha-ras genotype is lethal. Hemizygotes are selected by polymerase chain reaction (PCR) analysis of tail tips after birth. Spontaneous tumors in hemizygous transgenic mice are rare until 6 months of age. The observed rasH2 tumor spectrum, including lung adenoma/adenocarcinoma, forestomach and skin papillomas, Harderian gland adenoma, liver proliferative lesions, splenic hemangioma/sarcoma, and lymphoma is consistent with the BALB/c and C57BL/6 background. In the rasH2 mouse, point mutations of the transgene induced by genotoxins are reported frequently but not in all tumors. Elevated levels of transgene expression were detected in all genotoxin-induced tumors in the rasH2. Increased transgene expression was independent of the mutation rate in transgenic and endogenous ras genes. These observations suggest that the overexpression of transgenic c-Ha-ras is responsible for accelerated tumor development.

Developmental characterization and chromosomal mapping of a LacZ transgene expressed in the mouse apical ectodermal ridge.

The apical ectodermal ridge (AER) has an essential role in limb morphogenesis involving the specification of the proximal-distal axis of the limb. During the analysis of transgenic mice that harbor a LacZ transgene, we detected strong expression of beta-galactosidase within the AER of developing embryos. In this mouse line, called Z16, the bacterial LacZ gene is linked to a Herpes simplex virus immediate early promoter that is normally silent in mice. Embryos from other independent mouse lines harboring the same DNA construct exhibited no AER specific staining. Thus, it appears that the LacZ transgene in the Z16 line is expressed in the AER in response to regulatory influences from genomic DNA flanking the integration site. By fluorescent in situ hybridization, the transgene insertion site was mapped to chromosome 12. Hemizygous and homozygous transgenic mice appear normal and are fertile. AER specific beta-galactosidase staining was detected by 9.5 days post coitum in the forelimb and hindlimb bud. beta-galactosidase staining could be seen throughout the development of the limbs up to 14.5 days post coitum when expression was restricted to the distal-most regions of the digits of the hindlimbs. The loss of beta-galactosidase staining between digits correlated with the onset of programmed cell death, or apoptosis, in the digit interzones. LacZ expression in this transgenic line represents a useful marker for studying AER function in limb specification during mouse embryogenesis.