A cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells

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.     

Engineering mammalian chromosomes

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha- satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.      

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 micro-dissection 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 3–4 cM 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.This revised version was published online in November 2005 with corrections to the Cover Date.     

Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ~1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.     

Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA

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.     

Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously

Yeast artificial chromosomes (YACs) containing or lacking a biochemically defined DNA replication origin were transferred from yeast to mammalian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enable selection for YACs in mammalian cells and for monitoring YAC abundance in individual mammalian cells. All of eight clones made with linear and circularized YACs lacking the origin and seven of nine clones made with linear and circularized YACs containing the origin region contained single copies of the transfected YAC, along with various amounts of yeast DNA, integrated into single but different chromosomal sites. By contrast, two transformants derived from circularized YACs containing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many generations of in vitro propagation. Analysis of both clones at an early time after fusion revealed variously sized extrachromosomal YAC/yeast structures reminiscent of the extrachromosomal elements found in some cells harboring amplified genes. The data are consistent with the interpretation that YACs containing a biochemically defined origin of replication can initially replicate autonomously, followed by integration into multiple chromosomal locations, as has been reported to occur in many examples of gene amplification in mammalian cells.     

An integrated physical map for the short arm of human chromosome 5

The short arm of human chromosome 5 contains approximately 48 Mb of DNA and comprises 1.5% of the genome. We have constructed a mega-YAC/ STS map of this region that includes 436 YACs anchored by 216 STSs. By combining and integrating our map with the 5p maps of other groups using the same recombinant DNA library, a comprehensive map was constructed that includes 552 YACs and 504 markers. The YAC map covers >94% of 5p in four YAC contigs, bridges the centromere, and includes an additional 5 Mb of 5q DNA. The average marker density is 95 kb. This integrated 5p map will serve as a resource for the continuing localization of genes on the short arm of human chromosome 5 and as a framework for both generating and aligning the DNA sequence of this region.     

Addition of functional human telomeres to YACs

Linear mammalian artificial chromosomes (MACs) will requirefunctional telomeres, a centromere and the abllity to repllcateautonomously. We are investigating the possibility of developingMACs from yeast artificial chromosomes (YACs). Retrofittingvectors have been constructed to replace YAC telomeres withcloned human telomeric DNA. A modified YAC was introduced intomammalian cells by spheroplast tusion and the frequency withwhich the retrofitted human telomeric DNA seeded the formationof a new telomere was determined by Bal31 digestion and cytogeneticanalysis. The telomere adjacent to the selectable marker genewas functional in 5/46 clones (11%) while the telomere 200 kbaway at the other end of the YAC was functional in 1/46 clones(2%). These results indicate that despite the in vivo modificationof the end of the telomere by the addition of yeast sequences,human telomeres will function at a high enough frequency toallow the construction of MACs by this route.     

A high-resolution physical map of human chromosome 21p using yeast artificial chromosomes

The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes. Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region. We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs). To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs). Thirty YACs were selected from the St. Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries. These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12. The total size of the region covered by YACs is estimated between 2.9 and 5 Mb. The integrity of the YAC contig was confirmed by restriction enzyme fingerprinting and fluorescence in situ hybridization (FISH). One gap with an estimated size of 400 kb remained near the telomeric end of the contig. This YAC contig map of the short arm of human chromosome 21 constitutes a basic framework for further structural and functional studies of chromosome 21p.     

Integration of physical, breakpoint and genetic maps of chromosome 22. Localization of 587 yeast artificial chromosomes with 238 mapped markers

Detailed physical maps of the human genome are important resourcesfor the Identification and isolation of disease genes and forstudying the structure and function of the genome. We used datafrom STS content mapping of YACs and natural and induced chromosomalbreakpoints to anchor contigs of overlapping yeast artificialchromosome (YAC) clones spanning extensive regions of humanchromosome 22. The STSs were assigned to specific regions (bins)on the chromosome using cell lines from a somatic hybrid mappingpanel defining a maximum of 25 intervals. YAC libraries werescreened by PCR amplification of hierarchical pools of yeastDNA with 238 markers, and a total of 587 YAC clones were identified.These YACs were assembled into contigs based upon their sharedSTS content using a simulated annealing algorithm. Fifteen contigs,containing between 2 and 74 STSs were assembled, and orderedalong the chromosome based upon the cytogenetic breakpoint,meiotic and PFG maps. Additional singleton YACs were assignedto unique chromosomal bins. These ordered YAC contigs will beuseful for identifying disease genes and chromosomal breakpointsby positional cloning and will provide the foundation for higherresolution physical maps for large scale sequencing of the chromosome.     

Human mini-chromosomes with minimal centromeres

We have introduced a 6.5 Mb human mini-chromosome with a complex centromere structure into DT40 cells and have used sequence targeting and telomere-directed chromosome breakage to dissect the sequence requirements for centromere function. These experiments proved that a vertebrate centromere with two blocks of functional alphoid DNA separated by 2.5 Mb can exist as a stable structure in some but not all vertebrate cells. Further experiments indicated that recovery of chromosomes with less than approximately 100 kb of alphoid DNA is very inefficient, suggesting that a functional centromere requires a minimum of approximately 100 kb of alphoid DNA. Mini-chromosomes with minimal centromeres segregate accurately in some but not all vertebrate cells and should be useful for the detection of sequence-specific factors required for vertebrate centromere maintenance.     

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.     

Integration of physical, genetic and cytogenetic maps of human chromosome 7: isolation and analysis of yeast artificial chromosome clones for 117 mapped genetic markers

An Important goal of the human genome project is to assemblefully integrated physical, genetic and cytogenetic maps foreach human chromosome. Towards that end, we have isolated yeastartificial chromosome (YAC) clones containing 117 of the 119genetic markers that constitute a recently constructed, detailedgenetic map of human chromosome 7. Analysis of these clonesreveals numerous examples where adjacent genetic markers havebeen physically connected, either In Individual YACs or in multi-YACcontigs. At present, the 117 genetic markers are contained Infewer than 80 YAC contigs, with most of these contigs uniquelyordered relative to one another based on the genetic map positionsof the corresponding markers. These YAC and YAC contigs areestimated to contain {small tilde}60–85% of the DNA fromhuman chromosome 7. YACs representing 36 genetic markers weremapped by fluorescence In situ hybridization (FISH) to metaphasechromosomes, allowing assignment of these genetic markers tocytogenetic bands along chromosome 7 and placement of the centromerewithin the genetic map. Together, these studies provide geneticallyand cytogenetically anchored YAC clones covering the majorityof chromosome 7 that will be useful both for the positionalcloning of genes and as a framework for assembling a completeYAC-based physical map of the chromosome.     

A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region.

A contiguous set of 43 overlapping yeast artificial chromosome (YAC) clones has been developed for the Charcot-Marie-Tooth disease type 1A (CMT1A) duplication region of chromosome 17p11.2. The contig spans approximately 2.0 Mb and can be represented in a minimum of five overlapping YACs. The YAC clones were isolated from two total human genomic YAC libraries and from YAC libraries made from rodent-human hybrid cell lines. YAC clones were isolated from the libraries by polymerase chain reaction (PCR) technique. Localization to chromosome 17p11.2 was confirmed by fluorescence in situ hybridization. Overlap between the YAC clones was detected by inter-Alu PCR amplification of the YACs and by cross hybridization of the YACs with YAC insert ends obtained by Vectorette PCR. This YAC contig is a useful resource for analyzing and mapping all the genes contained within the CMT1A duplication.     

Cloning of the Huntington disease region in yeast artificial chromosomes.

The gene responsible for Huntington disease has been localized to a 2.5 million base pair (Mb) region between the loci D4S10 and D4S168 on the short arm of chromosome 4. As part of a strategy to clone the HD gene on the basis of its chromosomal location, we isolated genomic DNA from the HD region as a set of overlapping yeast artificial chromosome (YAC) clones. Twenty-eight YAC clones were identified by screening human YAC libraries with twelve PCR-based sequence-tagged sites (STSs) from the region. We assembled the YAC clones into overlapping sets by hybridizing them to a large number of DNA probes from the HD region, including the STSs. In addition, we isolated the ends of the human DNA inserts of most of the YAC clones to assist in the construction of the contig. Although almost half of the YACs appear to contain chimeric inserts and several contain internal deletions or other rearrangements, we were able to obtain over 2.2 Mb of the HD region in YACs, including one continuous segment of 2.0 Mb covering the region that most likely contains the HD gene. Ten of the twenty eight YAC clones comprise a minimal set spanning the 2.2 Mb. These clones provide reagents for the complete characterization of this region of the genome and for the eventual isolation of the HD gene.     

The major centromeric array of alphoid satellite DNA on the human Y chromosome is non-palindromic

We have determined the orientation of the major centromericalphoid array on the human Y chromosome. A PCR assay was usedto analyse the vector-insert junctions of seven YAC clones previouslypositioned on two independent Y chromosomes. The orientationis the same at all 10 positions measured. This suggests thatthe alphoid array is a simple unidirectional repeat throughoutand that human centromere structure thus differs from the palindromicorganisation found in Schizosaccharomyces pombe.     

The role of CENP-B and α-satellite DNA: de novo assembly and epigenetic maintenance of human centromeres

The centromere is an essential functional domain responsible for the correct inheritance of eukaryotic chromosomes during cell division. Eukaryotic centromeres include the highly conserved centromere-specific histone H3 variant, CENP-A, which has provided a powerful tool for investigating the recruitment of centromere components. However, the trigger that targets CENP-A to a specific genomic locus during centromere assembly remains unknown. Although, on rare occasions, CENP-A chromatin may assemble at non-centromeric DNA, all normal human centromeres are assembled and maintained on alpha-satellite (alphoid) DNA. The importance of alphoid DNA and CENP-B binding sites (CENP-B boxes), typical of normal human centromere DNA configurations, has been demonstrated through their requirement in de novo centromere assembly and Human Artificial Chromosome (HAC) assays. Mechanisms to link the centromere tightly to specific genomic sequences exist in humans and the two yeast species.     

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes

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.      

Localization of the 8;13 translocation breakpoint associated with myeloproliferative disease to a 1.5 mbp region of chromosome 13

There are five reported cases of an atypical myeloproliferative disorder in which the leukemia cells have a consistent t(8;13)(p11;q12) translocation. We analyzed the breakpoint in metaphases from two of these patients by fluorescence in situ hybridization using a series of yeast artificial chromosomes (YACs) derived from the 13q12 region. We found that a YAC containing the FLT1 and FLT3 oncogenes was localized distal to the 13q12 breakpoint and was not rearranged. YAC 66, a YAC that lies immediately adjacent to the chromosome 13 centromere, was localized proximal to the 13q12 breakpoint and was not rearranged. A third YAC, which is located between FLT1 and YAC 66, was unrearranged in normal metaphase chromosomes, but showed hybridization signals on both derivative chromosomes in both cases. Thus, the breakpoints in these two cases are localized to the same 1.5 Mbp region of 13q12. This may be the site of an unidentified gene involved in the pathogenesis of some types of leukemia.     

The Norrie disease gene maps to a 150 kb region on chromosome Xp11.3.

Norrie disease is a human X-linked recessive disorder of unknown etiology characterized by congenital blindness, sensory neural deafness and mental retardation. This disease gene was previously linked to the DXS7 (L1.28) locus and the MAO genes in band Xp11.3. We report here fine physical mapping of the obligate region containing the Norrie disease gene (NDP) defined by a recombination and by the smallest submicroscopic chromosomal deletion associated with Norrie disease identified to date. Analysis, using in addition two overlapping YAC clones from this region, allowed orientation of the MAOA and MAOB genes in a 5'-3'-3'-5' configuration. A recombination event between a (GT)n polymorphism in intron 2 of the MAOB gene and the NDP locus, in a family previously reported to have a recombination between DXS7 and NDP, delineates a flanking marker telomeric to this disease gene. An anonymous DNA probe, dc12, present in one of the YACs and in a patient with a submicroscopic deletion which includes MAOA and MAOB but not L1.28, serves as a flanking marker centromeric to the disease gene. An Alu-PCR fragment from the right arm of the MAO YAC (YMAO.AluR) is not deleted in this patient and also delineates the centromeric extent of the obligate disease region. The apparent order of these loci is telomere ... DXS7-MAOA-MAOB-NDP-dc12-YMAO.AluR ... centromere. Together these data define the obligate region containing the NDP gene to a chromosomal segment less than 150 kb.