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1.
We have shown previously that the tyrosinase gene encompassed in a 250 kb yeast artificial chromosome (YAC) is expressed faithfully in transgenic mice. To define the sequences important for this qualitatively and quantitatively correct expression pattern, we have generated transgenic mice with YACs carrying several deletions in the mouse tyrosinase locus. In particular, we wanted to address the in vivo relevance of a regulatory element indicated by a cell-specific DNase I hypersensitive site (HS) located -12 kb upstream of the gene. Wild-type level expression was observed only when the YACs transferred contained this HS. Constructs in which the HS was deleted gave rise to much weaker expression and variable patterns of expression. In conclusion, this HS region appears to harbour the essential regulatory element for the correct expression of the tyrosinase gene. Moreover, it behaves as a locus control region in that it commands the functional status of this expression domain, protecting it from position effects. 相似文献
2.
Kumud Majumder William Shawlot Gabriele Schuster Wilbur Harrison Frederick F.B. Elder Paul A. Overbeek 《Mammalian genome》1998,9(11):863-868
Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED).
On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial
chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10. To determine which of the
YACs contain the dl gene, we generated YAC transgenic mice by mouse embryo microinjections. The 200-kb YAC B25.D9 was found to rescue all of
the downless defects. In addition, the transgenic YAC rescued the dominant Sleek (Dl
slk
) allele. Since the sequences within the YAC are entirely deleted in one of the transgenic mutants, our results establish
that Sleek encodes a dominant-negative protein whose effects can be reversed by expression of extra copies of the wild-type
locus.
Received: 26 June 1998 / Accepted: 17 July 1998 相似文献
3.
Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression
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Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes. 相似文献
4.
Size Matters: Use of YACs,BACs and PACs in Transgenic Animals 总被引:15,自引:0,他引:15
In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences. 相似文献
5.
Gottfried Brem Urban Besenfelder Bernhard Aigner Mathias Müller Ines Liebl Guenther Schütz Lluís Montoliu 《Molecular reproduction and development》1996,44(1):56-62
The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis—such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene—have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation. © 1996 Wiley-Liss, Inc. 相似文献
6.
Moreira PN Pozueta J Pérez-Crespo M Valdivieso F Gutiérrez-Adán A Montoliu L 《Transgenic research》2007,16(2):163-168
Transgenes included in genomic-type constructs, such as yeast artificial chromosomes (YAC), P1-derived artificial chromosomes,
or bacterial artificial chromosomes (BAC), are normally correctly expressed, according to the endogenous expression pattern
of the homologous locus, because their large size usually ensures the inclusion of all regulatory elements required for proper
gene expression. The use of these large genomic-type transgenes is therefore the method of choice to overcome most position
effects, commonly associated with standard-type transgenes, and to guarantee the faithful transgene expression. However, in
spite of the different methods available, including pronuclear microinjection and the use of embryonic stem cells as vehicles
for genomic transgenes, the generation of transgenic animals with BACs and, particularly, with YACs can be demanding, because
of the low efficiencies requiring extensive microinjection sessions and/or higher number of oocytes. Recently, we have explored
the use of intracytoplasmic sperm injection (ICSI) into metaphase II oocytes as an alternative method for the generation of
YAC transgenic mice. Our results suggest that the use of transgenic strategies based on ICSI significantly enhances the efficiency
of YAC transgenesis by at least one order of magnitude. 相似文献
7.
Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells.
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Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC. 相似文献
8.
Peterson KR 《Expert reviews in molecular medicine》2003,5(13):1-25
The generation of transgenic mice with yeast artificial chromosomes (YACs) has proven to be a valuable system to: (1) study gene structure-function relationships; (2) produce mouse models of human disease; (3) complement mouse mutants; (4) generate mice bioreactors; and (5) screen YAC libraries in vivo. Continued refinement of current techniques and development of new protocols should encourage widespread adaptation of this strategy for these and other applications. Use of whole loci as transgenes is an important improvement in murine transgenesis because it results in a more realistic pattern and level of gene expression during ontogeny. Application of this technology to develop human artificial chromosomes (HACs) might provide the next generation of gene therapy vectors that will overcome most of the problems and barriers associated with current vector systems. 相似文献
9.
10.
A method for linking any standard yeast artificial chromosomes (YAC) is described. YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially. The linking vectors also contain the beta-galactosidase gene as an expression reporter in mammalian cells. 相似文献
11.
Libraries of the entire human genome, or regions of the genome, have been made in bacteria, yeast, and somatic cells. We have expanded this strategy using overlapping YACs and P1s from human 21q22.2 (the Down syndrome region) to create a panel of transgenic mice containing DNA that encompasses this region of the human genome. Together the members of the in vivo library, each with a unique transgene (four YACs and four P1s), contain approximately 2 Mb of contiguous DNA. The integrity, stable inheritance, and expression of a coding sequence for each member of the YAC panel are demonstrated, and the uses of the panel are described. 相似文献
12.
Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes. 总被引:12,自引:2,他引:12
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A Gnirke T S Barnes D Patterson D Schild T Featherstone M V Olson 《The EMBO journal》1991,10(7):1629-1634
Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC. 相似文献
13.
Retrofitting YACs for direct DNA transfer into plant cells 总被引:3,自引:0,他引:3
Gerhard Adam Jeffrey A. Mullen Karen L. Kindle 《The Plant journal : for cell and molecular biology》1997,11(6):1349-1358
The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures. 相似文献
14.
Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region 总被引:8,自引:0,他引:8
The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster. 相似文献
15.
《Genetic analysis, techniques and applications》1993,10(6):123-127
During the construction of yeast artificial chromosome (YAC) libraries to facilitate mapping of the human genome, two YACs may be cotransformed into the same yeast cell, making further analysis very difficult. We present a simple method to rescue the required YAC that utilizes the segregation of chromosomes at meiosis. In brief, we crossed the cotransformed yeast cell with a non-YAC-containing strain and induced the resulting diploid to sporulate and undergo meiosis. The new haploid generation included some yeast cells that contained only the desired YAC. These YACs were analyzed by conventional methods. To exclude the possibility that major rearrangement occurred during the procedure, we analyzed the YACs with restriction enzymes that cut only rarely. We conclude that this is a useful technique to rescue cotransformed YACs. 相似文献
16.
Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination 总被引:1,自引:0,他引:1
Douglas R. Smith Adrienne P. Smyth William M. Strauss Donald T. Moir 《Mammalian genome》1993,4(3):141-147
We have developed a pair of vectors for exchanging yeast artificial chromosome (YAC) arms by targeted homologous recombination. These conversion vectors allow the introduction of copy-number control elements into YACs constructed with pYAC4 or related vectors. YACs modified in this way provide an enriched source of DNA for genetic or biochemical studies. A LYS2 gene on the conversion vector provides a genetic selection for the modified YACs after transformation with appropriately prepared vector. A background of Lys+ clones that do not contain modified YACs is also present. However, clones with converted YACs can be distinguished from this background by counter-screening for loss of the original p YAC4 TRP1 arm (Trp- phenotype). The elimination of yeast replication origins (ARS elements) from the conversion vectors increased the frequency of Lys+ Trp- clones, but resulted in weaker amplification. Several YACs have been converted with these vectors, and the fate of the transformed DNA and of the resident YAC DNA has been systematically investigated. 相似文献
17.
Karin M. L. Gaensler Margit Burmeister Bernard H. Brownstein Patricia Taillon-Miller Richard M. Myers 《Genomics》1991,10(4)
The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human β-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult β-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire β-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the β-globin gene cluster. 相似文献
18.
We previously described the construction and characterization of aChlamydomonasgenomic library in yeast artificial chromosomes (YACs). Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on theunichromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI. Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads. This promises to make chromosomal walking inChlamydomonasa relatively efficient enterprise. We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA. The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most anyChlamydomonasgene. 相似文献
19.
Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast (YACs). 总被引:7,自引:1,他引:6
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Mammalian DNAs cloned as artificial chromosomes in yeast (YACs) frequently are chimeras formed between noncontiguous DNAs. Using pairs of human and mouse YACs we examined the contribution of recombination during transformation or subsequent mitotic growth to chimeric YAC formation. The DNA from pairs of yeast strains containing homologous or heterologous YACs was transformed into a third strain under conditions typical for the development of YAC libraries. One YAC was selected and the presence of the second was then determined. Co-penetration of large molecules, as deduced from co-transformation of markers identifying the different YACs, was > 50%. In approximately half the cells receiving two homologous YACs, the YACs had undergone recombination. Co-transformation depends on recombination since it was reduced nearly 10-fold when the YACs were heterologous. While mitotic recombination between homologous YACs is nearly 100-fold higher than for yeast chromosomes, the level is still much lower than observed during transformation. To investigate the role of commonly occurring Alu repeats in chimera formation, spheroplasts were transformed with various human YACs and an unselected DNA fragment containing an Alu at one end and a telomere at the other. When unbroken YACs were used, between 1 and 6% of the selected YACs could incorporate the fragment as compared to 49% when the YACs were broken. We propose that Alu's or other commonly occurring repeats could be an important source of chimeric YACs. Since the frequency of chimeras formed between YACs or a YAC and an Alu-containing fragment was reduced when a rad52 mutant was the recipient and since intra-YAC deletions are reduced, rad52 and possibly other recombination-deficient mutants are expected to be useful for YAC library development. 相似文献
20.
Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection 总被引:5,自引:0,他引:5
Moreira PN Giraldo P Cozar P Pozueta J Jiménez A Montoliu L Gutiérrez-Adán A 《Biology of reproduction》2004,71(6):1943-1947
The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI. 相似文献