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C Huxley

Publications and source records attributed to C Huxley.

At least 37 records · Page 2Linked to original sources
PubMed1999

In vivo analysis of DNase I hypersensitive sites in the human CFTR gene.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression. The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region. Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron. MATERIALS AND METHODS: In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice. The 310 kb human CFTR YAC was shown to restore the pheno-type of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR. RESULTS: We found that the YAC does not include the -79.5 kb region. The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature. The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression. The intron I DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands. CONCLUSION: These data support the in vivo importance of these regulatory elements.

Animals↗
PubMed1998

Exploring gene function: use of yeast artificial chromosome transgenesis.

Transgenesis is a very powerful tool in functional analysis of proteins and control of gene expression. One of the main drawbacks has been the low levels of expression, lack of tissue specificity, and inappropriate expression frequently observed for transgenes made with small plasmid-based constructs. The use of much larger DNA fragments cloned in yeast artificial clones (YACs), bacterial artificial clones, or P1-based artificial clones has been found to give much better levels of expression, generally very close to that of an endogenous gene, and tissue-specific expression matching that of the endogenous gene. In addition, the large DNA can easily be subtly modified by homologous recombination. This article describes the background and methods of YAC transgenesis.

Animals↗
PubMed1998

Correlation between varying levels of PMP22 expression and the degree of demyelination and reduction in nerve conduction velocity in transgenic mice.

Charcot-Marie-Tooth disease type 1A is most commonly caused by a duplication of a 1.5 Mb region of chromosome 17 which includes the peripheral myelin protein 22 gene (PMP22). Over-expression of this gene leads to a hypomyelinating/demyelinating neuropathy and to severely reduced nerve conduction velocity. Previous mouse and rat models have had relatively high levels of expression of the mouse or human PMP22 gene leading to severe demyelination. Here we describe five lines of transgenic mice carrying increasing copies of the human PMP22 gene (one to seven) and expressing increasing levels of the transgene. From histological and electrophysiological observations there appears to be a threshold below which expression of PMP22 has virtually no effect; below a ratio of human/mouse mRNA expression of approximately 0.8, little effect is observed. Between a ratio of 0.8 and 1.5, histological and nerve conduction velocity abnormalities are observed, but there are no behavioural signs of neuropathy. An expression ratio >1.5 leads to a severe neuropathy. A second observation concerns the histology of the different lines; the level of expression does not affect the type of demyelination, but influences the severity of involvement.

Animals↗
PubMed1998

The primary and final effector mechanisms required for kinin-induced epithelial chloride secretion.

The short-circuit current technique was used to examine the effects of N2-L-lysylbradykinin (LBK) on chloride secretion in the mucosae of the mouse intestine. It was found to be a potent chloride secretagogue in the mucosa lining the colon, jejunum, and cecum, as it is in most mammals, with 2 nM being sufficient to cause half-maximal secretion. The extent of the responses was in the order cecum > colon > jejunum. In cystic fibrosis (CF) null mice, with no CF transmembrane conductance regulator (CFTR) chloride channels, LBK caused no chloride secretion, but transporting activities for other ions were revealed. Introduction of the human CF gene into the genome of CF null mice at the zygote stage restored the chloride secretory activity of LBK, with only minor differences in potency. In mice in which the kinin B2 receptor gene had been disrupted, LBK had no effect, whereas the responses to forskolin were unchanged. Thus the acute effects of kinins on chloride secretion depend uniquely on kinin B2 receptors and CFTR chloride channels, which form the primary and final effector mechanisms of the secretory process.

Animals↗
PubMed1997

A dicistronic construct allows easy detection of human CFTR expression from YAC DNA in human cells.

We have made a dicistronic construct where the picornaviral internal ribosome-entry site (IRES) driving the expression of the beta-geo gene has been inserted into the 3'untranslated region of the human CFTR gene present in a YAC. When introduced into the human cell line Caco-2 expressing the CFTR gene, the expression of the dicistronic gene can be detected by lacZ staining and follows the accumulation of the endogenous CFTR mRNA upon differentiation of the cells. These data demonstrate that this IRES-based approach presents an alternative to mRNA in situ hybridisation and allows detection of expression in an autologous system.

Adenocarcinoma↗
PubMed1997

Contiguous arrays of satellites 1, 3, and beta form a 1.5-Mb domain on chromosome 22p.

The centromeric heterochromatin of all the human chromosomes is composed of megabases of tandemly repeated satellite DNA. Some of these sequences have been implicated in centromere formation and/or segregation but the arrangement of most of them on a large scale remains largely uncharacterized because of the difficulties in analyzing repetitive DNA. The alpha satellite is the best studied and is present in large tandem arrays at all centromeres, but satellites 1, 3, and beta have also been detected on a number of chromosomes. Here we have used FISH to extended DNA fibers to analyze these satellites on the short arm of the acrocentric chromosome 22. The satellite sequences were found to form a continuous domain spanning about 1.5 Mb and consisting of a major block of satellite 1 flanked by two blocks of beta satellite and three blocks of satellite 3. These six blocks of satellite DNA appear to form contiguous arrays with little intervening DNA.

Centromere↗
PubMed1997

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.

Animals↗
PubMed1997

Isolation of DNA from the centromere of human chromosome 7 by microdissection.

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a microdissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3-4cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.

Amniotic Fluid↗
PubMed1997

Copy number-dependent expression of a YAC-cloned human CFTR gene in a human epithelial cell line.

Initial studies for gene therapy of cystic fibrosis have used heterologous promoters to drive expression of the cystic fibrosis transmembrane regulator (CFTR) cDNA. An alternative approach would be to have constructs based on the endogenous promoter which could give tissue-specific and normal, regulated levels of expression. The DNA elements necessary for this remain unidentified so we have investigated CFTR expression from a 310 kb yeast artificial chromosome (YAC) clone which contains the intact human CFTR gene consisting of 27 exons spread over 230 kb of genomic DNA and also about 50 kb of 5' DNA. In order to distinguish the gene on the YAC, a restriction site change was introduced into the 3' untranslated region of the gene in the YAC. The YAC was then transferred into the human colonic adenocarcinoma cell line Caco-2 which expresses endogenous CFTR. The CFTR gene on the YAC was found to be well expressed in the Caco-2 cells and, in the two cell lines analysed, the ratio of YAC-derived mRNA to endogenous mRNA was the same as the ratio of YAC DNA to endogenous DNA. This indicates that each copy of the CFTR gene on a YAC integrated in the Caco-2 cells is being expressed at approximately the same level as each of the endogenous genes. Thus, the YAC clone contains all the long-range elements necessary to confer full levels of expression, independent of position of integration, in human Caco-2 cells. Deletion of this YAC clone should allow the elucidation of the DNA elements controlling human CFTR expression and the development of constructs for gene therapy.

Caco-2 Cells↗
PubMed1997

Benign cranial mediastinal lesions in three cats.

Cranial mediastinal lesions were detected in three cats, associated with respiratory impairment (case one), spontaneous pneumothorax (case two) and myasthenia gravis (case three), respectively. On gross and histological examination, the first case was considered either a lymphangioma or a branchial cystic mass of the thymic region of the mediastinum; a cystic lesion was suggested by sonographic detection of multiple anechoic cavitations within a circumscribed mass, while fine needle aspiration cytology excluded lymphosarcoma. The second case was diagnosed histologically as a cystic thymoma, but the third case was not examined microscopically. The masses were amenable to surgical excision in the first two cats, while this proved unnecessary in the third case because of resolution following treatment with dexamethasone. Corticosteroid responsiveness was unhelpful in distinguishing between these benign lesions and lymphosarcoma, as in two cases there was a partial or complete response to dosing with prednisolone or dexamethasone. These cases are presented to emphasise that conditions other than lymphosarcoma can produce cranial mediastinal lesions in cats, and that the prognosis for surgical treatment of lymphangiomas, multilocular thymic cysts and cystic thymomas can be excellent.

Animals↗
PubMed1997

Analysis of ribosomal and alphoid repetitive DNA by fiber-FISH.

We have used fluorescence in situ hybridization to extended DNA fibers (fiber-FISH) to analyse the alpha-satellite and rDNA arrays on a human chromosome 22. The rDNA could be seen as a string of signals spanning about 430 kb. The bulk of the alphoid DNA was found in a single large array of about 2.6 Mb.

Cell Line↗
PubMed1996

A shuttle system for transfer of YACs between yeast and mammalian cells.

The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.

Cell Fusion↗
PubMed1996

Replication of yeast DNA and novel chromosome formation in mouse cells.

To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells. Most of the lines contained stably integrated yeast DNA. However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection. This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation. In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere. The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration. These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA. Hence our results suggest caution be exercised in the interpretation of such assays.

Animals↗
PubMed1996

Removal and restoration of epithelial chloride secretory activity of kinins by gene manipulation.

Kinins are known to stimulate electrogenic chloride secretion in many mammalian epithelia, including those of the airways and the alimentary tract. In this study the chloride secretory activity of lysylbradykinin (LBK) on murine colonic epithelium has been examined, specifically to discover the primary and final effector mechanisms in this process, i.e., which kinin receptors are involved and which chloride channels are responsible for chloride secretion. The approach used was to modify the mice genetically and assess the effects on kinin mediated chloride secretion using voltage clamping at zero potential. Briefly, LBK increased SCC in mouse colon by approximately 150 microA cm-2 with an EC50 of approximately 5 nM. In null CF mice LBK, 1 microM had no effect on chloride secretion, but reduced SCC due to K+ secretion. This effect is normally masked in wild-type tissues by dominant chloride secretion, but can be shown to occur to the same extent by measuring K+ secretion with radioisotopes. Null CF mice produce no cftr, but CFTR was introduced into CF mice by injecting a YAC containing the human CF gene into the pronucleus of CF zygotes. Colonic epithelia from mice with the incorporated YAC showed the same sensitivity to LBK as wild-type tissues and achieved the same maximal chloride secretory response. Colonic epithelia from mice in which the B2r gene had been disrupted showed no response to LBK at normally supramaximally effective concentrations, although responses to other secretagogues were normal. Similarly des-Arg-BK caused no acute chloride secretory response in colonic epithelia from B2 knockout mice, however small responses appeared if tissues were incubated in vitro for 3-6 h. It is concluded that cftr chloride channels and B2rs are required for electrogenic chloride secretion. Further CFTR can replace cftr with no effect on either the sensitivity or extent of chloride secretion. In vitro, colonic epithelia may generate B1rs which, upon activation, have a minor effect on chloride secretory activity.

Animals↗
PubMed1996

Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA.

Construction of animal models of human inherited diseases is particularly important for testing gene therapy approaches. Towards this end, we constructed a mouse model for Charcot-Marie-Tooth disease type 1A by pronuclear injection of a YAC containing the human PMP22 gene. In one transgenic line, the YAC DNA is integrated in about eight copies and the PMP22 gene is strongly expressed to give a peripheral neuropathy closely resembling the human pathology. The disorder is dominant, causes progressive weakness of the hind legs, and there is severe demyelination in the peripheral nervous system including the presence of onion bulb formations. This approach will be valuable for pathologies produced by over-expression of a gene including trisomy and amplification in cancer. Such models will be particularly useful for testing gene therapy approaches if the transgene is human.

Animals↗