共查询到20条相似文献,搜索用时 31 毫秒
1.
Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes 总被引:1,自引:1,他引:1
Mogayzel PJ Jr; Henning KA; Bittner ML; Novotny EA; Schwiebert EM; Guggino WB; Jiang Y; Rosenfeld MA 《Human molecular genetics》1997,6(1):59-68
The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes
a transmembrane protein (CFTR) which functions in part as a cyclic
adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression
is controlled temporally and cell specifically by mechanisms that are
poorly understood. Insight into CFTR regulation could be facilitated by the
successful introduction of the entire 230 kb human CFTR and adjacent
sequences into mammalian cells. To this end, we have introduced two
different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620
kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing
human CFTR were identified by PCR, and the genetic and functional analyses
of one clone containing each YAC are described. Integration of the human
CFTR-containing YACs into the CHO genome at a unique site in each cell line
was demonstrated by fluorescence in situ hybridization (FISH). Southern
blot analysis suggested that on the order of one copy of human CFTR was
integrated per CHO cell genome. Fiber-FISH and restriction analysis
suggested that CFTR remained grossly intact. Northern analysis showed
full-length, human CFTR mRNA. Immunoprecipitation followed by
phosphorylation with protein kinase demonstrated mature, glycosylated CFTR.
Finally, chloride secretion in response to cAMP indicated the functional
nature of the human CFTR. This study provides several novel results
including: (i) functional human CFTR can be expressed from these YACs; (ii)
CHO cells are a permissive environment for expression of human CFTR; (iii)
the level of human CFTR expression in CHO cells is unexpectedly high given
the lack of endogenous CFTR production; and (iv) the suggestion by
Fiber-FISH of CFTR integrity correlates with functional gene expression.
These YACs and the cell lines derived from them should be useful tools for
the study of CFTR expression.
相似文献
2.
3.
4.
Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability 总被引:1,自引:15,他引:1
La Spada AR; Peterson KR; Meadows SA; McClain ME; Jeng G; Chmelar RS; Haugen HA; Chen K; Singer MJ; Moore D; Trask BJ; Fischbeck KH; Clegg CH; McKnight GS 《Human molecular genetics》1998,7(6):959-967
X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG
repeat expansion in the first exon of the androgen receptor (AR) gene.
Disease-associated alleles (37-66 CAGs) change in length when transmitted
from parents to offspring, with a significantly greater tendency to shift
size when inherited paternally. As transgenic mice carrying human AR cDNAs
with 45 and 66 CAG repeats do not display repeat instability, we attempted
to model trinucleotide repeat instability by generating transgenic mice
with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions
in their genomic context. Studies of independent lines of AR YAC transgenic
mice with CAG 45 alleles reveal intergenerational instability at an overall
rate of approximately 10%. We also find that the 45 CAG repeat tracts are
significantly more unstable with maternal transmission and as the
transmitting mother ages. Of all the CAG/CTG repeat transgenic mice
produced to date the AR YAC CAG 45 mice are unstable with the smallest
trinucleotide repeat mutations, suggesting that the length threshold for
repeat instability in the mouse may be lowered by including the appropriate
flanking human DNA sequences. By sequence-tagged site content analysis and
long range mapping we determined that one unstable transgenic line has
integrated an approximately 70 kb segment of the AR locus due to
fragmentation of the AR YAC. Identification of the cis - acting elements
that permit CAG tract instability and the trans -acting factors that
modulate repeat instability in the AR YAC CAG 45 mice may provide insights
into the molecular basis of trinucleotide repeat instability in humans.
相似文献
5.
Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA 总被引:4,自引:0,他引:4
DNA of two yeast artificial chromosomes (YACs) containing selectable human genes was transferred by microinjection to rodent cells in tissue culture. The human hypoxanthine phosphoribosyltransferase (HPRT) gene, spanning 45 kb, is contained on the 660-kb YAC yHPRT as described elsewhere. The human phosphoribosylglycinamide formyltransferase (GART) gene, spanning approximately 40 kb, is contained on the 590-kb YAC yGART2 as described previously. YAC DNA was isolated from pulsed-field gels and microinjected into mammalian cells in which the human HPRT and GART genes can be selected. The cell lines that were selected contain the entire human genes. Some of the cell lines contain multiple copies of the genes integrated at the same chromosomal position. The YAC yGART2 could not be purified away from natural yeast chromosomes of similar size, and the cell lines into which the human GART gene was introduced contain variable amounts of yeast DNA in addition to the human DNA. 相似文献
6.
7.
The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number. A targeting system was developed for the directed integration of circular YACs into mouse ES cells. The system combines Cre-lox recombination technology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene. Three independent germline competent ES cell lines [lox-containing ES lines (designated LES)] were created that contain a '-neo-lox' cassette integrated at different sites within the ES genome. A plasmid containing YAC vector sequences and a complementary '-neo-lox' cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21. The circularized YACs were then targeted to the lox sites of the LES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC. This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome. 相似文献
8.
(Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features 总被引:4,自引:0,他引:4
Peier AM McIlwain KL Kenneson A Warren ST Paylor R Nelson DL 《Human molecular genetics》2000,9(8):1145-1159
Fragile X syndrome is a common cause of mental retardation involving loss of expression of the FMR1 gene. The role of FMR1 remains undetermined but the protein appears to be involved in RNA metabolism. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macroorchidism and behavioral abnormalities. As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. We observed that the YAC transgene supported production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism was absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, we performed a more thorough evaluation of the Fmr1 knockout phenotype using additional behavioral assays that had not previously been reported for this animal model. The mouse displayed reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein did produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype. 相似文献
9.
Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA 总被引:2,自引:2,他引:2
Rozmahel R; Gyomorey K; Plyte S; Nguyen V; Wilschanski M; Durie P; Bear CE; Tsui LC 《Human molecular genetics》1997,6(7):1153-1162
We have used a mouse model to study the ability of human CFTR to correct
the defect in mice deficient of the endogenous protein. In this model,
expression of the endogenous Cftr gene was disrupted and replaced with a
human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for
the gene replacement failed to show neither improved intestinal pathology
nor survival when compared to mice completely lacking CFTR. RNA analyses
showed that the human CFTR sequence was transcribed from the targeted
allele in the respiratory and intestinal epithelial cells. Furthermore, in
vivo potential difference measurements showed that basal CFTR chloride
channel activity was present in the apical membranes of both nasal and
rectal epithelial cells in all homozygous knock-in animals examined. Ussing
chamber studies showed, however, that the cAMP-mediated chloride channel
function was impaired in the intestinal tract among the majority of
homozygous knock-in animals. Hence, failure to correct the intestinal
pathology associated with loss of endogenous CFTR was related to
inefficient functional expression of the human protein in mice. These
results emphasize the need to understand the tissue- specific expression
and regulation of CFTR function when animal models are used in gene therapy
studies.
相似文献
10.
11.
12.
Deborah E. Cabin Katheleen Gardiner Roger H. Reeves 《Somatic Cell and Molecular Genetics》1996,22(3):167-175
The mousePcp4 gene is highly expressed in brain, primarily in cerebellar Purkinje cells. It maps to chromosome 16 (Chr 16), in a region
of conserved synteny with human chromosome 21 (Chr 21). To further characterizePCP4 and its possible contribution to cerebellar hypoplasia in trisomy 21, or Down Syndrome (DS), we cloned and sequenced the
full length human cDNA, isolated a YAC which carries the entire gene, determined the gene structure, and characterized its
expression. The gene spans at least 55 kb and contains two introns, the placement of which is the same in mouse. Expression
in the mouse brain during development was detected at embryonic day 10, and thereafter through development. ThePCP4 YAC was placed on the human Chr 21 YAC contig by a link to a YAC carrying the markersD21S15 andD21S349. This placement distal toETS2 was confirmed by mapping on a somatic cell hybrid panel of Chr 21 translocations. This position caused an apparent break
in gene order with mouse Chr 16. However, mapping in the mouse was reassessed, andPcp4 and a linked marker,D16Mit71, were both moved distal toEts2, corresponding to the position ofPCP4 on Chr 21. 相似文献
13.
Initiation of DNA replication from within the Epstein-Barr virus (EBV) latent cycle origin oriP occurs once per cell cycle and is almost entirely dependent upon cellular proteins. The human origin recognition complex (ORC) is recruited to oriP and orchestrates the events that lead to the initiation of replication. EBNA-1, the sole viral protein required for oriP-plasmid replication, binds four sites within the replicator but the role(s) it plays in the replication of oriP plasmids has not been elucidated. We investigated the recruitment of ORC to oriP in vivo and show that the binding of EBNA-1 to the replicator is necessary for the association of the ORC subunit Orc2 with the replicator. The minimal replicator of oriP consists of two EBNA-1 binding sites flanked by perfect 14-bp inverted repeats (a and b), but these repeats are dispensable for the association of Orc2 with the replicator. A mutational analysis of the 14-bp repeats provided additional support for a role for the telomere repeat binding protein 2 in oriP replicator function. We show that nucleotide differences between the oriP replicator of the B95-8 and Raji EBV genomes are not solely responsible for the inefficient utilization of this origin in the Raji EBV genome. 相似文献
14.
The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component of oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication. 相似文献
15.
16.
17.
Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype 总被引:4,自引:4,他引:4
Hodgson JG; Smith DJ; McCutcheon K; Koide HB; Nishiyama K; Dinulos MB; Stevens ME; Bissada N; Nasir J; Kanazawa I; Disteche CM; Rubin EM; Hayden MR 《Human molecular genetics》1996,5(12):1875-1885
Huntington disease (HD) is caused by expansion of a CAG trinucleotide
repeat in exon 1 of a novel gene. The HD protein (huntingtin) plays a
critical role in early embryonic development since homozygous targeted
disruption of the murine HD gene results in embryonic lethality by day 7.5.
To rescue this phenotype by transgene based huntingtin expression it is
therefore essential to express the protein early enough in development in
the appropriate cells. Since YAC based transgenes are known to be regulated
in an appropriate temporal and tissue-specific manner, we sought to rescue
the embryonic lethality by breeding YAC transgenic mice expressing human
huntingtin with mice heterozygous for the targeted disruption. We generated
viable offspring homozygous for the disrupted murine HD gene but expressing
human huntingtin derived from the YAC. This result clearly shows that YAC
transgene based expression of huntingtin occurs prior to 7.5 days
gestation. Additionally, we show that human huntingtin expression in YAC
transgenic mice follows an identical tissue distribution and subcellular
localisation pattern as that of the murine endogenous protein and that
expression levels of 2-3 times endogenous can be achieved. This shows that
human huntingtin under the influence of its native promoter, despite
differences to the murine protein, is functional in a murine background and
can compensate for loss of the murine protein. These results show that YAC
transgenic approaches are a particularly promising route to producing an
animal model for disorders associated with CAG expansion.
相似文献
18.
Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.
The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG)(n)repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA. 相似文献
19.
Functional screening and complex traits: human 21q22.2 sequences affecting learning in mice 总被引:4,自引:0,他引:4
Libraries of the mammalian genome have generally been propagated in single
cells and have been used for gene discovery through in vitro analyses. We
have expanded upon this concept by the creation of panels of YAC transgenic
mice propagating targeted megabase regions of the genome. Such a panel of
mice can be called an 'in vivo library' and genes can be identified based
on functional screens of members of the library. To test this approach, we
created a 2 Mb in vivo library of human chromosome 21q22.2. Analysis of the
library has revealed that one 570 kb YAC, in two separate founder lines,
was associated with distinct learning deficits compared with the other
21q22 YAC transgenics and non- transgenic control animals. We have
localized the gene on the YAC that causes the deficits by taking advantage
of fragmentation of the YAC during the process of microinjection. The
responsible gene is the human minibrain gene, and the homolog of the gene
in Drosophila is also associated with learning defects. These results
suggest that altered dosage of minibrain is associated with abnormal neural
development in flies and mice and, in humans, may also be involved in the
molecular pathology of Down syndrome.
相似文献
20.
De novo formation of several features of a centromere following introduction of a Y alphoid YAC into mammalian cells 总被引:9,自引:1,他引:8
The DNA sequence requirements for mammalian centromere functionhave been Investigated by re-Introducing human YAC clones containingeither centromeric or non-centromeric sequences Into hamsterand human cells. All YACs integrated into the host chromosomes.In most cell lines produced by spheroplast fusion into hamstercells, intact copies of the YAC and a large amount of yeastDNA were found. Cell lines produced by lipofection Into humancells usually contained simple structures without yeast DNA.YACs containing Y alphoid DNA reformed several of the propertiesof a centromere, Including a cytogenetlcally visible constriction,CREST antiserum binding and disruption of anaphase chromosomemovement. In contrast, YACs containing non-centromeric sequencesproduced none of these results. This work suggests that a fewhundred kb of alphoid DNA is sufficient to reconstitute severalImportant features of a centromere. 相似文献