Human Yeast Artificial Chromosome Research Articles

Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast–embryonic stem cell fusion

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

Complementation of a Defect in the Asparagine-Linked Glycosylation of a Mouse FM3A Mutant G258 Cell Line by Spheroplast Fusion of a Human Mega YAC Clone 923f5

Mouse G258 mutant stopped both cell growth and the synthesis of lipid-linked oligosaccharide at the Man(3)GlcNAc(2)-P-P-Dolichol at a restricted temperature with a single gene mutation. To clarify the lesion in the G258 mutant, we isolated human genomic DNA transformants of the G258 mutant, which recovered from both defects by way of cell hybridization with X-ray irradiated HeLa cells. We detected a common 1.3-kb product by inter-human specific sequence in the L1 (L1Hs) PCR in the transformants (Kataoka et al., Somat. Cell Mol. Genet., 24, 235-243 (1998)). In the present study, we screened a human mega yeast artificial chromosome (YAC) library by PCR with primers designed according to the 1.3-kb DNA, and selected YAC clone 923f5. Moreover, we found by spheroplast fusion that YAC clone 923f5 complemented both defects of the G258 mutant. Since the human counterpart of the yeast ALG11 gene is localized in the region, the G258 mutant might have a defect in the mouse ALG11 gene.

Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor

Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest—a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo r), replaced the α-lactalbumin gene in a 210 kb human α-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock.

A Human Yeast Artificial Chromosome Containing the Multiple Endocrine Neoplasia Type 2B Ret Mutation Does Not Induce Medullary Thyroid Carcinoma but Does Support the Growth of Kidneys and Partially Rescues Enteric Nervous System Development in Ret-Deficient Mice

We generated a line of transgenic mice using a yeast artificial chromosome containing the Ret mutation responsible for the multiple endocrine neoplasia type 2B syndrome (MEN 2B). The resulting animals did not develop any of the expected neoplasms associated with MEN 2B. Transgenic animals were then bred with animals lacking murine Ret (Ret(M)) to further evaluate the function of human mutated Ret (Ret(H)(2B)) in the murine context. Whereas mice lacking Ret(M) exhibit intestinal aganglionosis and the absence of kidneys with other genitourinary anomalies, expression of the Ret(H)(2B) transgene in Ret(M)-deficient mice allowed significant renal development with a partial rescue of the enteric nervous system. These Ret(H)(2B)-positive/Ret(M)-deficient mice exhibit normal Ret expression and survive longer than Ret(M)-deficient mice, but still die at 3 to 5 days of age with evidence of enterocolitis. We conclude that the normal expression of a human Ret proto-oncogene with the MEN 2B mutation does not cause any features of MEN 2B in mice. Although the gene is normally expressed in the appropriate target tissues, there is incomplete phenotypic rescue in mice lacking murine Ret. These results suggest important interspecies differences between humans and mice in the function of the Ret oncogene.

Defining a metabolic phenotype in the brain of a transgenic mouse model of spinocerebellar ataxia 3.

Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.

Rescue of the lethal scl−/− phenotype by the human SCL locus

The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in the development of both blood and endothelium. Loss-of-function studies have shown that SCL is essential for the formation of hematopoietic stem cells, for subsequent erythroid development and for yolk sac angiogenesis. SCL exhibits a highly conserved pattern of expression from mammals to teleost fish. Several murine SCL enhancers have been identified, each of which directs reporter gene expression in vivo to a subdomain of the normal SCL expression pattern. However, regulatory elements necessary for SCL expression in erythroid cells remain to be identified and the size of the chromosomal domain needed to support appropriate SCL transcription is unknown. Here we demonstrate that a 130-kilobase (kb) yeast artificial chromosome (YAC) containing the human SCL locus completely rescued the embryonic lethal phenotype of scl(-/-) mice. Rescued YAC(+) scl(-/-) mice were born in appropriate Mendelian ratios, were healthy and fertile, and exhibited no detectable abnormality of yolk sac, fetal liver, or adult hematopoiesis. The human SCL protein can therefore substitute for its murine homologue. In addition, our results demonstrate that the human SCL YAC contains the chromosomal domain necessary to direct expression to the erythroid lineage and to all other tissues in which SCL performs a nonredundant essential function.

YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebellar deficit.

Machado-Joseph disease (MJD; MIM 109150) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the MJD1 gene. We have previously reported the generation of human yeast artificial chromosome (YAC) constructs encompassing the MJD1 locus into which expanded (CAG)(76) and (CAG)(84) repeat motifs have been introduced by homologous recombination. Transgenic mice containing pathological alleles with polyglutamine tract lengths of 64, 67, 72, 76 and 84 repeats, as well as the wild type with 15 repeats, have now been generated using these YAC constructs. The mice with expanded alleles demonstrate a mild and slowly progressive cerebellar deficit, manifesting as early as 4 weeks of age. As the disease progresses, pelvic elevation becomes markedly flattened, accompanied by hypotonia, and motor and sensory loss. Neuronal intranuclear inclusion (NII) formation and cell loss is prominent in the pontine and dentate nuclei, with variable cell loss in other regions of the cerebellum from 4 weeks of age. Interestingly, peripheral nerve demyelination and axonal loss is detected in symptomatic mice from 26 weeks of age. In contrast, transgenic mice carrying the wild-type (CAG)(15) allele of the MJD1 locus appear completely normal at 20 months. Disease severity increases with the level of expression of the expanded protein and the size of the repeat. These mice are representative of MJD and will be a valuable resource for the detailed analysis of the roles of repeat length, tissue specificity and level of expression in the neurodegenerative processes underlying MJD pathogenesis.

Precise mapping of breakpoints in conserved synteny between human chromosome 1 and pig chromosomes 4, 6 and 9.

Previous comparative mapping suggested that at least five pig chromosomes (Sscr4, 6, 9, 10 and 14) share homology with human chromosome 1 (Hsap1). A significant quantitative trait loci (QTL) for fat deposition has been identified on Sscr4 that appears to be near the junction region between Sscr4 and Sscr9 relative to Hsap1. It is of interest to define the boundaries of conserved synteny between pig chromosomes and Hsap1 to use human map information to identify putative comparative positional candidates for this QTL. Eleven genes, including Janus kinase 1 (JAK1), Prostaglandin E receptor3 (PTGER3), urate oxidase (UOX), coagulation factor 3 (F3), vascular cell adhesion molecule 1 (VCAM1), ribosomal protein L5 (RPL5), POU domain, class 2, transcription factor 1 (POU2F1), coagulation factor 5 (F5), Prostaglandin endoperoxide synthase-2 (PTGS2), myosin binding protein H (MYBPH) and Antithrombin III (SERPINC1), were selected to refine the boundaries of the blocks of conserved synteny between Hsap1 and pig chromosomes. Pig sequence tagged sites (STSs) were developed and used to physically map these 11 genes using a somatic cell hybrid panel. Eight loci have been mapped by using fluorescent in situ hybridization (FISH) to improve map resolution. Heterologous FISH was used to refine the location of VCAM1 on human chromosomes. In addition, human yeast artificial chromosomes (YACs) were mapped by heterologous FISH on pig metaphases to refine the boundaries of the regions of homology between Sscr4 and Sscr9 on Hsap1. Results from this study suggest the precise break in conserved synteny on Hsap1 corresponding to the Sscr4/6 and Sscr4/9 transitions are most likely on the Hsap1p22 and Hsap1q24-25 regions, respectively. Further, our data predict that Hsap1q21-24 is a candidate region for the backfat QTL localized to Sscr4.

A Yeast Artificial Chromosome Containing the Human Preprotachykinin-A Gene Expresses Substance P in Mice and Drives Appropriate Marker-Gene Expression during Early Brain Embryogenesis

We have produced a yeast artificial chromosome (YAC) transgenic model containing the human preprotachykinin-A gene (hPPTA) that can drive appropriate expression of β-galactosidase within the adult mouse brain. Here, we investigate its embryonic expression to assess the transcriptional regulation of the PPTA gene during the development of several neural pathways later affected by disease in humans. We demonstrate that the human PPTA gene regulatory region is active in appropriate areas of the developing brain at significantly earlier time points than has been previously reported. Furthermore, despite replacement of most of the 3′ untranslated region by the marker gene cassette, the modified human YAC is able to express substance P (SP) on a murine SP/NKA−/− background. This transgenic model, in addition to being valuable in examining the hPPTA regulatory region, will also prove to be important in exploring the downstream function of the gene in the adult and the embryo brain.

A human YAC transgene rescues craniofacial and neural tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth.

The platelet-derived growth factor alpha-receptor (PDGFRalpha) plays a vital role in the development of vertebrate embryos, since mice lacking PDGFRalpha die in mid-gestation. PDGFRalpha is expressed in several types of migratory progenitor cells in the embryo including cranial neural crest cells, lung smooth muscle progenitors and oligodendrocyte progenitors. To study PDGFRalpha gene regulation and function during development, we generated transgenic mice by pronuclear injection of a 380 kb yeast artificial chromosome (YAC) containing the human PDGFRalpha gene. The YAC transgene was expressed in neural crest cells, rescued the profound craniofacial abnormalities and spina bifida observed in PDGFRalpha knockout mice and prolonged survival until birth. The ultimate cause of death was respiratory failure due to a defect in lung growth, stemming from failure of the transgene to be expressed correctly in lung smooth muscle progenitors. However, the YAC transgene was expressed faithfully in oligodendrocyte progenitors, which was not previously observed with plasmid-based transgenes containing only upstream PDGFRalpha control sequences. Our data illustrate the complexity of PDGFRalpha genetic control, provide clues to the location of critical regulatory elements and reveal a requirement for PDGF signalling in prenatal lung growth, which is distinct from the known requirement in postnatal alveogenesis. In addition, we found that the YAC transgene did not prolong survival of Patch mutant mice, indicating that genetic defects outside the PDGFRalpha locus contribute to the early embryonic lethality of Patch mice.

The Human Preprotachykinin-A Gene Promoter Has Been Highly Conserved and Can Drive Human-like Marker Gene Expression in the Adult Mouse CNS

Toward an understanding of the mechanisms controlling Preprotachykinin-A (PPTA) transcription, we introduced a 380-kb human yeast artificial chromosome containing the PPTA gene tagged with the β-galactosidase gene into transgenic mice. This resulted in a pattern of LacZ expression in the central nervous system (CNS) remarkably similar to that reported for PPTA mRNA in the rat. However, the human gene drove expression in areas of the mouse CNS not associated with strong PPTA expression in rodents but which have been shown to express PPTA in the human. This study clearly demonstrates the high degree of conservation of the mechanisms involved in PPTA transcription that has occurred throughout 100 million of divergent human and rodent evolution. This study also defines the maximum linear extent of the human PPT-A promoter. We believe these findings constitute the removal of a significant obstacle in studying the transcriptional regulation of the human PPTA gene in vivo.

Faithful expression of the human 5q31 cytokine cluster in transgenic mice.

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.

Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome.

Novel pseudogenes homologous to the mitochondrial (mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copy number polymorphism of the mtDNA pseudogenes was observed among randomly chosen individuals, and even among siblings. A mtDNA pseudogene in the Y-chromosome was observed in a YAC clone carrying only repetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showed that there were at least 5.7 x 10(5) bp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part of the human nuclear genome is discussed.

In Vivo Analysis of DNase I Hypersensitive Sites in the Human CFTR Gene

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. 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. 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. These data support the in vivo importance of these regulatory elements.

Integrity of Human YACs during Propagation in Recombination-Deficient Yeast Strains

Several isogenic strains with defects in recombination/repair genes (RAD1, RAD50, RAD51, RAD52, RAD54,andRAD55) were examined for their ability to propagate accurately a variety of linear and circular yeast artificial chromosomes (YACs) containing human DNA inserts. To assess YAC stability, the human DNA inserts were internally marked by anADE2-pBR-URA3cassette. Following selection for Ura−clones on 5-fluoroorotic acid containing medium, the following types of YAC deletions were identified: (i) those caused by homologous recombination with a telomericpBRsequence; (ii) internal deletions, presumed to occur by recombination between commonly occurring DNA repeats such asAluandLINEsequences; and (iii) deletions leading to loss of part of a YAC arm.rad52host strains, but not other recombination-deficient strains, decreased the rate of all types of YAC deletions 25- to 400-fold. We have also developed and testedkar1strains with a conditionalRAD52gene that allow transfer of a YAC from any host into a recombination-deficient background. These strains provide an efficient tool for stabilization of YACs and are useful for allowing additional recombinational modification of YACs.

New applications of low-C0tDNA as a DNA fingerprint probe.

New applications of low-C0t DNA are reported as probes for genetic identification and genome characterization. These fast and intermediately reannealing fractions have sometimes either been discarded in genomic library construction to enhance the probability of finding single copy genes, or they are used as resources for identifying individual repetitive sequences. In addition, they are used as blockers to enhance hybridization signals. C0t-1 DNA serves as a probe for DNA fingerprinting of human yeast artificial chromosomes. We have isolated low-C0t DNA from bacteria, fungus, plant, mussel, chicken, rat and fish from the sheared genomic DNA of the respective species. Low-C0t DNA is labeled to generate DNA fingerprints and for in situ hybridization. Individual specific DNA fingerprint profiles are observed and species-specific DNA fragments can be identified in bacteria, fungus, plants (Ginseng and Amaranthus) and mussel. When low-C0t DNA probes from rat, chicken and fish were employed, only smear profiles and no distinct DNA banding patterns were evident. In these species, individual clones can be used as a probe for DNA fingerprinting containing repetitive sequences after subcloning. The advantage of this approach is to quickly develop a useful probe for DNA fingerprinting for genetic identification and analysis without sequencing knowledge a priori. This represents an innovative approach to the use of these repetitive components of the genome.

Construction of Human Chromosome 16- and 5-Specific Circular YAC/BAC Libraries byin VivoRecombination in Yeast (TAR Cloning)

Transformation-associated recombination (TAR) in yeast was exploited for the selective isolation of human DNAs as large circular yeast artificial chromosomes (YACs) from two rodent/human hybrid cell lines containing human chromosomes 5 and 16. TAR cloning vectors containing the F-factor origin of replication were constructed for use in these experiments. Presence of the F-factor origin in TAR vectors provides the capability of transferring the YACs generated byin vivorecombination in yeast intoEscherichia colicells and propagating them as bacterial artificial chromosomes (BACs). A high enrichment of human versus rodent YACs was observed during isolation of human DNA from the rodent/human hybrid cell lines. Although <3% of the DNA content in the hybrid cells was human, as many as 75% of the transformants contained human YACs. In contrast to the standard YAC cloning method based onin vitroligation, no human/mouse chimeras were observed during TAR cloning. The constructed human chromosome 16 YAC library had approximately 2.6× coverage, represented by 4320 YAC clones with an average insert size of 80 kb. YAC clones generated from chromosome 16 were successfully converted into BACs by electroporation of DNA isolated from yeast transformants intoE. coli.The BAC clones represent ∼0.6× chromosomal coverage. Pilot YAC and BAC libraries of chromosome 5 have been also constructed. The chromosomal distribution of YAC/BACs from chromosome 5 and chromosome 16 was evaluated by fluorescencein situhybridization (FISH). The distribution of FISH signals appeared random along the length of each chromosome. We conclude that TAR cloning provides an efficient means for generating representative chromosome-specific YAC/BAC libraries.

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR).

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR).

The Human Glutamate Receptor δ2 Gene (GRID2) Maps to Chromosome 4q22

We isolated the human glutamate receptor δ2 (GRID2) gene, which has 97.0% identity in amino acid sequence to the mouse glutamate receptor δ2 (Grid2) gene. We subsequently mapped this gene to human chromosome 4q22 by radiation hybrid mapping and by hybridization to two overlapping human yeast artificial chromosomes that are located in 4q22. TheGrid2gene, which is mutated in lurcher (Lc) mice, maps to mouse chromosome 6. Thus, the mapping of theGRID2gene to human chromosome 4q22 confirms and refines a region of synteny between mouse and human genomes.

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting.

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.