Independent Mouse Lines Research Articles

Constitutive expression of tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice.

Here we describe a new mouse model with constitutive expression of the catalytic subunit of telomerase (Tert) targeted to thymocytes and peripheral T cells (Lck-Tert mice). Two independent Lck-Tert mouse lines showed higher incidences of spontaneous T-cell lymphoma than the corresponding age-matched wild-type controls, indicating that constitutive expression of Tert promotes lymphoma. Interestingly, T-cell lymphomas in Lck-Tert mice were more disseminated than those in wild-type controls and affected both lymphoid and nonlymphoid tissues, while nonlymphoid tissues were never affected with lymphoma in age-matched wild-type controls. Importantly, these roles of Tert constitutive expression in promoting tumor progression and dissemination were independent of the role of telomerase in telomere length maintenance, since telomere length distributions on a single-cell basis were identical in Lck-Tert and wild-type thymocytes. Finally, Tert constitutive expression did not interfere with telomere capping in Lck-Tert primary thymocytes, although it resulted in greater chromosomal instability upon gamma irradiation in Lck-Tert primary lymphocytes than in controls, suggesting that Tert overexpression may interfere with the cellular response to DNA damage.

Inducible and selective transgene expression in murine vascular endothelium.

We have developed a system utilizing the murine Tie2 promoter/enhancer coupled with the "tetracycline-on" regulatory elements to create a model that allows regulated and selective expression of a beta-galactosidase (betaGal) reporter transgene in the adult murine vascular endothelium. Two independent lines of viable and fertile mice were characterized, and they exhibit minimal betaGal expression under basal conditions. In response to exogenous doxycycline (Dox), selective expression of betaGal was demonstrated in the vascular endothelium of all tissues examined. En face analyses of the aorta and its principle branches indicate that the vast majority of lumenal endothelial cells express the transgene. Inducible betaGal expression also extends to the endocardium and the microvasculature of all organs. There is no evidence of specific transgene expression in nonendothelial cell types. Induction of the betaGal was effectively achieved after 3 days of oral Dox treatment and persisted for over 3 mo with continuous administration. This model can now be widely applied to study the role of specific genes in the phenotype of adult murine vasculature.

Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures.

Persons affected with tuberous sclerosis complex (TSC) develop a wide range of neurological abnormalities including aberrant neuronal migration and seizures. In an effort to model TSC-associated central nervous system abnormalities in mice, we generated two independent lines of astrocyte-specific Tsc1 conditional knockout mice by using the Cre-LoxP system. Astrocyte-specific Tsc1-null mice exhibit electroencephalographically proven seizures after the first month of age and begin to die at 3 to 4 months. Tsc1-null mice show significant increases in astrocyte numbers throughout the brain by 3 weeks of age and abnormal neuronal organization in the hippocampus between 3 and 5 weeks. Moreover, cultured Tsc1-null astrocytes behave similar to wild-type astrocytes during log phase growth but demonstrate increased saturation density associated with reduced p27(Kip1) expression. Collectively, our results demonstrate that astrocyte-specific disruption of Tsc1 in mice provides a context-dependent growth advantage for astrocytes that results in abnormalities in neuronal organization and epilepsy.

Increased myocardial Rab GTPase expression: a consequence and cause of cardiomyopathy.

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing beta(2)-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose-dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) alpha and delta, and confocal microscopy showed abnormal subcellular distribution of PKCalpha in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.

A Mutant Form of JAB/SOCS1 Augments the Cytokine-induced JAK/STAT Pathway by Accelerating Degradation of Wild-type JAB/CIS Family Proteins through the SOCS-box

Cytokines exert biological functions by activating Janus tyrosine kinases (JAKs), and JAK inhibitors JAB (also referred to as SOCS1 and SSI1) and CIS3 (SOCS3) play an essential role in the negative regulation of cytokine signaling. We have found that transgenic (Tg) mice expressing a mutant JAB (F59D-JAB) exhibited a more potent STAT3 activation and a more severe colitis than did wild-type littermates after treatment with dextran sulfate sodium. We now find that there is a prolonged activation of JAKs and STATs in response to a number of cytokines in T cells from Tg mice with lck promoter-driven F59D-JAB. Overexpression of F59D-JAB also sustained activation of JAK2 in Ba/F3 cells. These data suggested that F59D-JAB up-regulated STAT activity by sustaining JAK activation. To elucidate molecular mechanisms related to F59D-JAB, we analyzed the effects of F59D-JAB on the JAK/STAT pathway using the 293 cell transient expression system. We found that the C-terminal SOCS-box played an essential role in augmenting cytokine signaling by F59D-JAB. The SOCS-box interacted with the Elongin BC complex, and this interaction stabilized JAB. F59D-JAB induced destabilization of wild-type JAB, whereas overexpression of Elongin BC canceled this effect. Levels of endogenous JAB and CIS3 in T cells from F59D-JAB Tg-mouse were lower than in wild-type mice. We propose that F59D-JAB destabilizes wild-type, endogenous JAB and CIS3 by chelating the Elongin BC complex, thereby sustaining JAK activation.

A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and beta-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the alpha-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, beta-galactosidase and immunochemical stainings revealed differences in GFP and beta-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense beta-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

ENU Mouse Mutagenesis: Generation of Mouse Mutants with Aberrant Plasma IgE Levels

Background: The ENU Mouse Mutagenesis Project aims at a large-scale, systematic production of mouse mutants using the alkylating agent ethyl-nitrosourea (ENU). Offspring of mutagenized mice are subjected to a multiparameter screen to detect alterations in various phenotypes with the ultimate goal of identifying novel genes relevant for the expression of the phenotype. Using this approach, we have analyzed plasma IgE concentrations to identify mouse mutants with aberrant plasma IgE levels. Methods and Results: ENU-mutagenized male C3HeB/FeJ were mated to wild-type females to produce F1 offspring. F1 animals were analyzed for alterations in their plasma IgE concentrations that showed a dominant mode of inheritance, or bred further to screen for recessive phenotypes. Plasma IgE concentrations were determined by ELISA and a normal range for plasma IgE was established using C3HeB/FeJ wild-type animals. So far we have tested 6568 F1 animals. Repeated testing confirmed a stable aberrant IgE phenotype in 124 animals. To confirm the genetic basis of the observed phenotype, these mice were subjected to confirmation crossing. Currently we have established 9 independent mutant mouse lines (3 with high plasma IgE and 6 with plasma IgE below detection limit) that have been genetically confirmed and additional 24 variant mouse lines are currently undergoing confirmation testing. Conclusion: ENU mouse mutagenesis allowed us to generate and identify mouse mutants with aberrant plasma IgE levels, which may be used to characterize novel genes involved in IgE regulation and may serve as animal models for IgE-mediated diseases.

Role of EP2and EP3PGE2receptors in control of murine renal hemodynamics

The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE(2) E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE(2). Specifically, we determined the extent to which the EP(2) and EP(3) receptor subtypes mediate the actions of PGE(2) on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP(2) or EP(3) (-/-) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP(2) receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP(2)-/- mice [RBF EP(2)-/-: 5.3 +/- 0.8 ml. min(-1). 100 g kidney wt(-1); renal vascular resistance (RVR) 19.7 +/- 3.6 mmHg. ml(-1). min. g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 +/- 0.5 ml. min(-1). 100 g kidney wt(-1); RVR +/+: 25.4 +/- 4.9 mmHg. ml(-1). min. 100 g kidney wt(-1)). This was also the case for the peak RBF increase after local PGE(2) (500 ng) injection into the renal artery (EP(2)-/-: 116 +/- 4 vs. +/+: 112 +/- 2% baseline RBF). In contrast, we found that the absence of EP(3) receptors in EP(3)-/- mice caused a significant increase (43%) in basal RBF (7.9 +/- 0.8 ml. min(-1). g kidney wt(-1), P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 +/- 1.4 mmHg. ml(-1). min. g kidney wt(-1), P < 0.05 vs. +/+). Local administration of 500 ng of PGE(2) into the renal artery caused more pronounced renal vasodilation in EP(3)-/- mice (128 +/- 2% of basal RBF, P < 0.05 vs. +/+). We conclude that EP(3 )receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP(3) receptors are capable of buffering PGE(2)-mediated renal vasodilation.

Functional Embryonic Cardiomyocytes after Disruption of the L-type α1C (Cav1.2) Calcium Channel Gene in the Mouse

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.

Dissociation of Direct and Indirect Effects of Angiotensin II on the Heart

33 Cardiac angiotensin II (Ang II), either derived from the circulation or locally synthesized, is often suggested to be involved in the structural adaptations occurring in the heart in hypertension and following myocardial infarction. However, it is debated whether the proven beneficial effects of renin-angiotensin system blockade in these pathologies are related to an inhibition of the direct cardiac actions of the peptide. The objective of the present study was to investigate which of the effects of cardiac Ang II are due to direct stimulation of cardiac cells by Ang II. To test for cardiac specific functions of Ang II, transgenic mice were developed that express an Ang II-releasing fusion protein (J Biol Chem 1997;272:12994-99) exclusively in cardiomyocytes. Blood pressure, heart rate, cardiac and plasma Ang II content, Ang II receptor binding and organ morphology were monitored in transgenic (TG) and non-transgenic littermate mice (control). Cardiac Ang II levels in TG mice were 20-40 fold higher than in hearts of control mice (15±3 pg/100 mg ww). In 3 independent founder lines of TG mice, plasma Ang II concentration was not altered as compared to control (119±20 vs. 127±20 pg/mL). The heart weight to body weight ratio in TG mice (4.0±0.1 mg/g) was not different from controls (3.8±0.1 mg/g), neither was systolic pressure (137±4 and 138±7 mm Hg respectively) or heart rate (618±13 and 662±15 bpm respectively). Microscopic inspection of TG hearts did not reveal any differences with control regarding size and number of cardiomyocytes and organization of extracellular matrix proteins. TG mice had not become less sensitive for Ang II signaling since Ang II receptor number was not altered in TG mice (Bmax = 23±3 fmol/mg protein) as compared to control (22±2 fmol/mg protein). Our data show that very high Ang II levels in hearts of TG mice do not lead to myocardial enlargement or affect cardiovascular physiology. We conclude that elevated Ang II in the heart has no direct effects on cardiac cells and we hypothesize that effects of cardiac Ang II become apparent upon altered hemodynamic loading.

The Development and Subsequent Elimination of Aberrant Peripheral Axon Projections in Semaphorin3A Null Mutant Mice

Semaphorin3A (previously known as Semaphorin III, Semaphorin D, or collapsin-1) is a member of the semaphorin gene family, many of which have been shown to guide axons during nervous system development. Semaphorin3A has been demonstrated to be a diffusible chemorepulsive molecule for axons of selected neuronal populations in vitro. Analysis of embryogenesis in two independent lines of Semaphorin3A knockout mice support the hypothesis that this molecule is an important guidance signal for neurons of the peripheral nervous system (M. Taniguchi et al., 1997, Neuron 19, 519–530; E. Ulupinar et al., 1999, Mol. Cell. Neurosci. 13, 281–292). Surprisingly, newborn Semaphorin3A null mutant mice exhibit no significant abnormalities (O. Behar et al., 1996, Nature 383, 525–528). In this study we have tested the hypothesis that guidance abnormalities that occurred during early stages of Semaphorin3A null mice development are corrected later in development. We have found that the extensive abnormalities formed during early developmental stages in the peripheral nervous system are largely eliminated by embryonic day 15.5. We demonstrate further that at least in one distinct anatomical location these abnormalities are mainly the result of aberrant projections. In conclusion, these findings suggest the existence of correction mechanisms that eliminate most sensory axon pathfinding errors early in development.

The Egr-1 promoter contains information for constitutive and inducible expression in transgenic mice.

Egr-1 is an immediate early gene that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene is expressed in many cell types and is induced by a wide variety of extracellular signals. The mechanisms by which the Egr-1 gene is regulated in vivo remain poorly understood. In this study, we have generated transgenic mice with a construct containing 1200 bp of the mouse Egr-1 promoter coupled to nuclear localized LacZ. In multiple independent lines of mice, reporter gene expression was detected in subsets of endothelial cells, vascular smooth-muscle cells, cardiomyocytes, neurons, and hepatocytes. This pattern closely resembled that of the endogenous gene. After partial hepatectomy, reporter gene activity was upregulated between two- and fivefold in regenerating livers. Taken together, these findings suggest that the Egr-1 promoter contains information for appropriate spatial and temporal expression in vivo.

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Specificity and Efficiency of Cre-Mediated Recombination in Emx1–cre Knock-in Mice

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles and its expression is restricted to the neurons in the developing and adult cerebral cortex and hippocampus. We reported previously the creation of a line of transgenic mice in which the cre gene was placed directly downstream of the putative Emx1 promoter using ES cell technology. We showed that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. In the present study, the specificity and efficiency of the cre-mediated recombination were determined using three independent lines of reporter mice and a combination of histochemical staining, neuronal culture, and Southern detection of the genomic DNA. Our results showed that the recombination was highly efficient in all three lines of reporter mice tested and confirmed that the deletion was restricted to the neurons in the cerebral cortex and hippocampus. Furthermore, we have determined that the recombination efficiency in the cerebral cortex was 91%. Our results suggest that Emx1 is not expressed in every neuron in the developing and adult cerebral cortex. This line of cre mice should contribute to the studies of cortical development and plasticity.

Prostaglandin D synthase gene is involved in the regulation of non-rapid eye movement sleep.

To examine the function of prostaglandin (PG) D synthase (PGDS) gene, as well as endogenously produced PGD(2) in sleep regulation in vivo, we generated transgenic (TG) mice that overexpress human PGDS gene to study their sleep behavior. Although no difference was observed in the sleep/wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye movement (NREM), but not in rapid eye movement (REM), sleep was observed in two independent lines of TG mice after stimulation by tail clipping. Concomitantly, the spontaneous locomotor activity of TG animals was drastically decreased in response to the tail clip. Induction of NREM sleep in TG mice was positively correlated with the PGD(2) production in the brain. Sleep, locomotion, and PGD(2) content were essentially unchanged in wild-type mice after tail clipping. The results with TG mice demonstrate the involvement of the PGDS gene in the regulation of NREM sleep.

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.

Transgenic mice overexpressing tartrate-resistant acid phosphatase exhibit an increased rate of bone turnover.

Tartrate-resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate-limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number-dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low-level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue-specific promoter.

Transgenic Analysis of a 100-kb Human β-Globin Cluster–Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome

AbstractTo date, the normal transcriptional regulation of the human β-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human β-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human β-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F1 mice showed that 2 lines express human β globin at levels approximately equivalent to the endogenous mouse β-major genes. One line expresses no human β globin, while the remaining 6 lines show intermediate expression levels. Complete γ→β-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic→adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.

Transgenic Analysis of a 100-kb Human β-Globin Cluster–Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome

To date, the normal transcriptional regulation of the human β-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human β-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human β-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F1 mice showed that 2 lines express human β globin at levels approximately equivalent to the endogenous mouse β-major genes. One line expresses no human β globin, while the remaining 6 lines show intermediate expression levels. Complete γ→β-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic→adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.

Tissue-specific transcriptional activity of a pancreatic islet cell-specific enhancer sequence/Pax6-binding site determined in normal adult tissues in vivo using transgenic mice.

A pancreatic islet cell-specific enhancer sequence (PISCES) shared by the rat insulin-I, glucagon, and somatostatin genes binds the paired domain-containing transcription factor Pax6 and confers strong transcriptional activity in pancreatic islet cell lines. It was found recently that Pax6 plays a major role in islet development. In the present study, transgenic mice were used to investigate PISCES-mediated transcription in normal adult islets in vivo. In several independent mouse lines expressing a PISCES-luciferase reporter transgene, the PISCES motif directed gene expression in the adult eye, cerebellum, and discrete brain areas, consistent with the tissue distribution of Pax6. These tissues contain two Pax6 isoforms caused by alternative splicing, only one of which was found to bind the PISCES motif in electrophoretic mobility shift assays. No reporter gene expression was detected in adult pancreatic islets or in any other peripheral organ tested. RT-PCR analysis confirmed that Pax6 mRNA is present in adult islets. These results demonstrate that the PISCES motif is sufficient to direct highly tissue-specific gene expression in whole animals. The lack of PISCES-mediated transcription in adult islets indicates that the Pax6 protein(s) expressed in adult pancreatic islets function differently from the ones in the eye and cerebellum.