Production of a severe cystic fibrosis mutation in mice by gene targeting.

We have used gene targeting in embryonic stem cells to introduce an HPRT mini-gene into the coding sequence of the murine cystic fibrosis gene (cftr). This insertion introduces a termination codon in frame with the cftr coding sequence to terminate prematurely the CFTR protein within the first nucleotide binding domain. Animals homozygous for the cftr disruption fail to thrive and display a range of symptoms including meconium ileus, distal intestinal obstructions, gastrointestinal mucus accumulation and blockage of pancreatic ducts. The animals also show lacrimal gland pathology. Tracheal and caecal transepithelial current measurements demonstrate the lack of a cAMP activatable Cl- channel. These animals will prove useful for the evaluation of new therapeutic drugs and gene therapy strategies.

Generation and characterization of a delta F508 cystic fibrosis mouse model.

We have generated mice carrying the most common mutation in cystic fibrosis (CF), delta F508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. delta F508-/- mice die from peritonitis and show deficiencies in cAMP-activated electrogenic Cl- transport. These mice produce delta F508 transcripts and show the temperature-dependent trafficking defect first described for the human delta F508 CFTR protein. A functional CFTR Cl- channel not demonstrated by null CF mice or present at 37 degrees C was detected following incubation of epithelial cells at 27 degrees C. Thus, these mice are an accurate delta F508 model and will be valuable for testing drugs aimed at overcoming the delta F508 trafficking defect.

The challenge of fetal gene therapy.

Three recent reports describe the first in vivo attempts at fetal gene therapy. The results underline the need for more intensive studies of the scientific and ethical implications of this new and perhaps more preventive approach to gene therapy.

Transgenic mouse models to study Gpr54/kisspeptin physiology.

Four transgenic mouse lines have been generated with mutations in the Gpr54 gene and two lines with mutations in the Kiss1 gene. In general, the phenotypes of all these mutant mice are very similar and provide evidence that these molecules constitute an authentic receptor/ligand pair with no obvious redundancy or overlap with other signaling pathways. The mutant mice all fail to undergo pubertal maturation and show poor development of the gonads and infertility with low sex steroid and gonadotrophic hormone levels (hypogonadotrophic hypogonadism). Spermatogenesis and ovulation are severely impaired and mutant females do not show estrous cycling. The gonads and the anterior pituitary retain functional responses to hormonal stimulation however, consistent with the primary defect being a failure to secrete gonadotrophin releasing hormone (GnRH) from the hypothalamus. Slight differences between the phenotype of some of the mutant lines may reflect the type of mutation carried by each line. These mutant mice are being used to interrogate the function of Gpr54 and Kiss1 in key aspects of mammalian reproduction in vivo including the role of these proteins in the generation of the pre-ovulatory luteinizing hormone (LH) surge and aspects of sexual behavior. They provide a useful resource to further understand the hypothalamic regulation of mammalian reproduction, its integration with the pituitary-gonadal axis and to study the potential function of Gpr54 and Kiss1 in peripheral tissues.

Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice.

Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero-injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (Cftr(tmlCam)) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr(-/-) mice treated with the CFTR-adenovirus and those treated with the control vector.

Cystic fibrosis gene therapy.

A variety of cystic fibrosis gene therapy approaches based on viral (adenovirus, retrovirus, and adeno-associated virus) and non-viral (liposomes and receptor-mediated endocytosis) routes are currently being assessed for safety and efficacy. Of these, the trials involving liposomal and adenoviral vectors are the most advanced, as both have been shown to correct the cystic fibrosis Cl- conductance defect in vivo.

Kisspeptin neurones in the posterodorsal medial amygdala modulate sexual partner preference and anxiety in male mice.

The posterodorsal medial amygdala (MePD) is a neural site in the limbic brain involved in regulating emotional and sexual behaviours. There is, however, limited information available on the specific neuronal cell type in the MePD functionally mediating these behaviours in rodents. The recent discovery of a significant kisspeptin neurone population in the MePD has raised interest in the possible role of kisspeptin and its cognate receptor in sexual behaviour. The present study therefore tested the hypothesis that the MePD kisspeptin neurone population is involved in regulating attraction towards opposite sex conspecifics, sexual behaviour, social interaction and the anxiety response by selectively stimulating these neurones using the novel pharmacosynthetic DREADDs (designer receptors exclusively activated by designer drugs) technique. Adult male Kiss-Cre mice received bilateral stereotaxic injections of a stimulatory DREADD viral construct (AAV-hSyn-DIO-hM3 D(Gq)-mCherry) targeted to the MePD, with subsequent activation by i.p. injection of clozapine-N-oxide (CNO). Socio-sexual behaviours were assessed in a counter-balanced fashion after i.p. injection of either saline or CNO (5 mg kg-1 ). Selective activation of MePD kisspeptin neurones by CNO significantly increased the time spent by male mice in investigating an oestrous female, as well as the duration of social interaction. Additionally, after CNO injection, the mice appeared less anxious, as indicated by a longer exploratory time in the open arms of the elevated plus maze. However, levels of copulatory behaviour were comparable between CNO and saline-treated controls. These data indicate that DREADD-induced activation of MePD kisspeptin neurones enhances both sexual partner preference in males and social interaction and also decreases anxiety, suggesting a key role played by MePD kisspeptin in sexual motivation and social behaviour.

Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen.

Site-directed mutagenesis was used to change Lys-128 of the simian virus 40 large-T nuclear location signal to Met, Ile, Arg, Gln, Asn, Leu, or His. Except for the large-T antigen of the Arg mutation, which was present in cytoplasmic and nuclear compartments, the resultant proteins were unable to enter the nucleus. By contrast, mutations at other sites within the signal were generally less severe in their effect. In some cases (Lys-128 to Gln, Asn, and His), the apparently cytoplasmic variants were able to support limited plasmid DNA replication, suggesting that low levels of large-T antigen undetectable by immunofluorescence were present in the nucleus. Such mutants did not support viral DNA replication. We conclude that there is a strong requirement for a basic residue at position 128 in the large-T nuclear location signal, with Lys the preferred residue.

Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice.

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.

Thymic lymphomas in mice with a truncating mutation in Brca2.

Inherited mutations in the BRCA2 gene predispose women to breast and ovarian cancer. We created a mutation in the mouse Brca2 gene that terminates translation in exon 11 at 45% of the normal transcript length. Ninety % of Brca2(tm1Cam) homozygous mutant mice die prenatally or perinatally. The location of the Brca2(tm1Cam) mutation differs from those reported previously, and this phenotype suggests a correlation with genotype analogous to that previously reported in humans. Although heterozygote mice have remained free of tumors for 10 months, Brca2(tm1Cam) homozygous mutants that survived to adulthood died with thymic lymphomas between 12 and 14 weeks of age.

Visualisation of Kiss1 Neurone Distribution Using a Kiss1-CRE Transgenic Mouse.

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE-GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss-CRE heterozygous mice with CRE-activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80-90% of tdTomato positive neurones in the ARC were co-labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato-labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre-optic areas and to the lateral hypothalamus.

Analysis of A-MuLV transformed fibroblast lines for amplification of the c-myc, p53 and c-fos nuclear proto-oncogenes.

Aberrations in nuclear proto-oncogene organisation and/or gene expression have been implicated in cell transformation mediated by the v-abl gene. For example, it has been suggested that amplification of the c-myc proto-oncogene is a co-operative event in v-abl induced fibroblast transformation. We have investigated amplification of the c-myc, p53 and c-fos nuclear proto-oncogenes in several Abelson murine leukaemia virus (A-MuLV) transformed fibroblast lines. None of these proto-oncogenes were detectably rearranged or amplified in v-abl transformed Swiss 3T3 lines. In contrast, NIH3T3 fibroblasts transformed by the v-abl gene consistently showed a 4 to 16-fold amplification of the c-myc gene. These data show that c-myc gene amplification is not an obligatory event associated with A-MuLV transformation, but may be restricted to cell lines derived from NIH3T3. c-myc gene amplification also did not correlate with a reduced latency period for tumour induction in nude mice. In addition, c-myc amplification was not selected during tumourigenesis, indicating that this event is not required for A-MuLV transformed Swiss 3T3 cells to display a full tumourigenic phenotype.

Search of the 5' untranslated region of the human cardiac actin gene for segments controlling translation.

We have examined the possibility that the 5'-UT region of the human cardiac actin (CH-actin) mRNA is responsible for regulating translation of this transcript during skeletal myogenesis. Genes were constructed which consisted of the murine leukaemia virus promoter driving the Escherichia coli LacZ coding region with and without the CH-actin 5'-UT region. These constructs were transfected into L6 myoblasts that were subsequently differentiated into myotubes. The presence of the CH-actin 5'-UT region appeared to have no effect on expression of the LacZ reporter gene. EGTA blocks myogenesis and inhibits translation of muscle-specific transcripts (Endo, T. and Nadal-Ginard, B. (1987) Cell 49, 515-526) but EGTA had no effect on expression of the chimaeric LacZ transcripts. Thus, if the CH-actin transcript is subject to translational regulation, it must be mediated by sequences other than those of the 5'-UT region.

Kiss1 mutant placentas show normal structure and function in the mouse.

INTRODUCTION: Kisspeptins, encoded by the Kiss1 gene, are a set of related neuropeptides that are required for activation of the mammalian reproductive axis at puberty and to maintain fertility. In addition, kisspeptin signaling via the G-protein coupled receptor GPR54 (KISS1R) has been suggested to regulate human placental formation and correlations have been found between altered kisspeptin levels in the maternal blood and the development of pre-eclampsia. METHODS: We have used Kiss1 and Gpr54 mutant mice to investigate the role of kisspeptin signaling in the structure and function of the mouse placenta. RESULTS: Expression of Kiss1 and Gpr54 was confirmed in the mouse placenta but no differences in birth weight were found in mice that had been supported by a mutant placenta during fetal development. Stereological measurements found no differences between Kiss1 mutant and wild-type placentas. Measurement of amino-acid and glucose transport across the Kiss1 mutant placentas at E15.5 days did not reveal any functional defects. DISCUSSION: These data indicate that mouse placentas can develop a normal structure and function without kisspeptin signaling and can support normal fetal development and growth.

Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice.

Multiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl-) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (delta PD) following addition of amiloride and subsequent removal of Cl- from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (-24.9 +/- 2.1 mV) than in normal mice (-6.3 +/- 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl- substitution (delta PD of 8.5 +/- 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl- or Na+ transport defects were observed when 5 x 10(9) IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5-20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (delta PD of 5.9 +/- 1.1 mV) to low Cl- substitution was detected 2 days later. In these mice, the basal nasal PD (-10.5 +/- 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+ transport defect. Expression of functional CFTR activity was transient with no measurable delta PD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+ and Cl- ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.

Markedly reduced effects of (-)-isoprenaline but not of (-)-CGP12177 and unchanged affinity of beta-blockers at Gly389-beta1-adrenoceptors compared to Arg389-beta1-adrenoceptors.

1. Substitution of arginine by glycine at position 389, a frequent beta(1)-adrenoceptor polymorphism, reduces adenylyl cyclase stimulation by (-)-isoprenaline. beta(1)-Adrenoceptors mediate the effects of catecholamines and nonconventional partial agonists ((-)-CGP12177) through different sites. We investigated the influence of the 389 polymorphism on beta blocker affinity, as well as on the responses to (-)-isoprenaline and the nonconventional partial agonist (-)-CGP12177 on cyclic AMP levels in CHO cells expressing recombinant Arg389-beta(1)-adrenoceptors (101 fmol mg(-1) protein) or Gly389-beta(1)-adrenoceptors (94 fmol mg(-1)). 2. The affinity of beta-blockers and partial agonists, estimated from competition binding with (-)-[(125)I]-cyanopindolol, was not different for Arg389-beta(1)-adrenoceptors and Gly389-beta(1)-adrenoceptors. 3. The maximum cAMP increases by (-)-isoprenaline and (-)-CGP12177 at Gly389-beta(1)-adrenoceptors were reduced by 97 and 46%, but the potencies enhanced 2 and 0.5 log units, respectively, compared to Arg389-beta(1)-adrenoceptors. The intrinsic activity of (-)-CGP12177 with respect to the (-)-isoprenaline was 0.057 at Arg389-beta(1)-adrenoceptors and 1.05 at Gly389-beta(1)-adrenoceptors. 4. We confirm in intact CHO cells that responses to (-)-isoprenaline are markedly reduced at Gly389-beta(1)-adrenoceptors compared to Arg389-beta(1)-adrenoceptors. However, the 389 polymorphism reduces considerably less the agonist responses to (-)-CGP12177, indicating that coupling to G(s) protein is different for beta(1)-adrenoceptors activated by catecholamines than for receptors activated by nonconventional partial agonists. The affinity of beta-blockers is conserved across the Arg389Gly polymorphism.

Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.

1. Guanylin, a 15 amino acid endogenous gut peptide, increased the short circuit current (SCC) in the epithelium of the mouse colon, but only when applied to the apical and not the basolateral surface. 2. By use of selective blockers of epithelial ion transport and modification of the bathing solution, it was concluded that guanylin increased electrogenic chloride secretion but also had a minor effect on electrogenic sodium absorption. In addition there were small residual currents which remained unresolved. 3. The threshold concentration of guanylin causing a SCC increase was less than 50 nM, but at concentrations 40 times greater no indication of a maximally effective concentration was found. 4. Two guanylin isomers with the same amino acid sequence but with the disulphide bridges joined in an alternate fashion showed no activity. Thus only guanylin with the greatest structural homology to heat stable enterotoxin (STa) showed biological activity. 5. The action of guanylin was virtually eliminated in colonic epithelia from transgenic cystic fibrosis (CF) mice. As these animals lack the chloride channel coded by the CF gene sequence, it is likely that the final effector process in murine colonic epithelia involves the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. 6. Opportunistic infections of the gut generating STa lead to diarrhoeal conditions via an action of the toxin on apical guanylin receptors. Thus, as discussed, the CF heterozygote may have a genetic advantage in this circumstance.

A comparison of topoisomerase I activity in normal and transformed cells.

Many viral oncogenes encode protein-tyrosine kinase activities. However, important in vivo substrates of these enzymes have yet to be identified. Recently, type I topoisomerases were shown to be in vitro substrates for two tyrosine kinases. Following tyrosine phosphorylation, topoisomerase I activity was reduced 10-fold (Tse-Dinh et al. Nature 312: 785-786, 1984). To determine whether topoisomerase I activity was modulated by tyrosine phosphorylation in vivo, we have measured topoisomerase I activity in nuclear lysates prepared from both normal fibroblasts and cells transformed by two different viral oncogenes (v-abl, v-src). Under a variety of experimental conditions, we have found no evidence to support the notion that type I topoisomerase activity is modulated by tyrosine phosphorylation in vivo.

Acetylcholine induces cytosolic Ca2+ mobilization in isolated distal colonic crypts from normal and cystic fibrosis mice.

In intestinal biopsies from cystic fibrosis (CF) patients acetylcholine fails to elicit a chloride secretion response, and this observation can be explained by a defect in the Ca2+ signalling pathway in CF secretory cells. We tested the hypothesis that in CF intestine, the generation of an intracellular Ca2+ signal upon cholinergic stimulation is absent. A transgenic CF mouse model was used. Electrical measurements on intact jejunum and unstripped colon were performed in Ussing chambers. Intact distal colonic crypts were isolated, and the intracellular Ca2+ concentration was monitored using the Ca2+-sensitive dye fura-2. Acetylcholine increased the short-circuit current generated by wild-type jejunum and colon, but failed to induce a response in CF tissues. Acetylcholine caused a transient elevation in the intracellular Ca2+ concentration in colonic crypts from both wild-type and CF mice; the amplitude and timing of the response in CF crypts was indistinguishable from that in wild-type crypts. The response to acetylcholine was also observed in the absence of extracellular calcium, indicating intracellular stores as the source from which the cytosolic Ca2+ concentration increased. We conclude that the absence of a cholinergically-induced secretory response in CF intestine is not due to a defect in the generation of a Ca2+ signal in intestinal cells upon cholinergic stimulation.