Transgenic mice carrying yeast artificial chromosomes

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.     

Preparation of intact yeast artificial chromosome DNA for transgenesis of mice

Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.     

Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.

We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.     

Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection

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.     

High-efficiency yeast artificial chromosome fragmentation vectors

Chromosome fragmentation vectors (CFVs) are used to create deletion derivatives of large fragments of human DNA cloned as yeast artificial chromosomes (YACs). CFVs target insertion of a telomere sequence into the YAC via homologous recombination with Alu repetitive elements. This event results in the loss of all YAC sequences distal to the site of integration. A new series of CFVs has been developed. These vectors target fragmentation to both Alu and LINE human repetitive DNA elements. Recovery of deletion derivatives is ten- to 20-fold more efficient with the new vectors than with those described previously.     

Unequal homologous recombination of human DNA on a yeast artificial chromosome.

We examined unequal homologous DNA recombination between human repetitive DNA elements located on a yeast artificial chromosome (YAC) and transforming plasmid molecules. A plasmid vector containing an Alu element, as well as a sequence identical to a unique site on a YAC, was introduced into yeast and double recombinant clones analyzed. Recombination occurs between vector and YAC Alu elements sharing as little as 74% identity. The physical proximity of an Alu element to the unique DNA segment appears to play a significant role in determining the frequency with which that element serves as a recombination substrate. In addition, cross-over points of the recombination reaction are largely confined to the ends of the repetitive element. Since a similar distribution of crossover sites occurs during unequal homologous recombination in human germ and somatic tissue, we propose that similar enzymatic processes may be responsible for the events observed in our system and in human cells. This suggests that further examination of the enzymology of unequal homologous recombination of human DNA within yeast may yield a greater understanding of the molecular events which control this process in higher eukaryotes.     

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.     

Simplified construction and characterization of yeast artificial chromosome libraries.

Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.     

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.     

An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.     

Generation of large insert yeast artificial chromosome libraries

The development of YAC cloning technology has directly enhanced the relationship among genetic, physical, and functional mapping of genomes. Because of their large size, YACs have enabled the rapid construction of physical maps by ordered clone mapping and contig building, and they complement other molecular approaches for mapping complex genomes. Large insert libraries are constructted by size fractionating large DNA embedded in agarose and protecting DNA from degradation with polyamines.     

Construction of a bovine yeast artificial chromosome (YAC) library

We have constructed a bovine yeast artificial chromosome (YAC) library to provide a common resource for bovine genome research. We used leukocytes of a Japanese black bull ( Bos taurus ) as the DNA source, AB1380 for the yeast host, and pYAC4 for the vector. The library consists of 24 576 clones arranged in 256 96-well microtiter plates. An average insert size estimated from the analysis of 251 randomly selected clones was 480 kb. The rate of chimeric YACs evaluated by fluorescence in situ hybridization (FISH) analysis of 44 randomly selected clones was 36·4%. To estimate the number of genome equivalents, PCR-based screening was performed with 48 primer pairs and isolated 3·2 clones on average. In order to provide broad access for the scientific community, this library has been incorporated into the Reference Library system which provides high density filters for colony hybridization screening and a common database of the library.     

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.