scholarly journals Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice

1993 ◽  
Vol 90 (23) ◽  
pp. 11381-11385 ◽  
Author(s):  
K M Gaensler ◽  
M Kitamura ◽  
Y W Kan
Keyword(s):  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Physiological Role ◽  
Artificial Chromosome ◽  
Chromatin Domain ◽  
Globin Genes ◽  
Beta Globin ◽  
Cis Acting ◽  
Artificial Chromosomes

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.

scholarly journals Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines

1993 ◽  
Vol 90 (23) ◽  
pp. 11207-11211 ◽  
Author(s):  
K R Peterson ◽  
G Zitnik ◽  
C Huxley ◽  
C H Lowrey ◽  
A Gnirke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Artificial Chromosome ◽  
Globin Genes ◽  
Beta Globin ◽  
Control Of Gene Expression ◽  
Artificial Chromosomes ◽  
Mouse Erythroleukemia

We demonstrate that transfer of a yeast artificial chromosome (YAC) containing 230 kb of the human beta-globin locus into mouse erythroleukemia cells by fusion results in correct developmental regulation of the human beta-like globin genes. Additionally, we show that early after hybrid formation, human embryonic epsilon- and fetal gamma-globin genes are coexpressed with the adult beta gene but that after 10-20 weeks in culture, globin gene expression switches to predominantly adult. Thus, in contrast to shorter gene constructs, the globin genes of the beta-globin locus YAC are regulated like the chromosomal globin genes. These results indicate that transfer of YACs into established cell lines can be used for the analysis of the developmental control of multigenic and developmentally regulated human loci.

Transgenic mice

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 938-949 ◽  
Author(s):  
C. Babinet ◽  
D. Morello ◽  
J. P. Renard
Keyword(s):  
Homologous Recombination ◽  
Transgenic Mice ◽  
Dna Sequences ◽  
Insertional Mutagenesis ◽  
Germ Line ◽  
Embryonic Stem ◽  
Physiological Role ◽  
Cis Acting ◽  
Endogenous Genes

Stable integration into the mouse genome of exogenous genetic information has become, over the past few years, a very potent approach for different aspects of biology. It is a common feature that the integrated exogenous gene (the transgene) is expressed properly both spatially and temporally. Constructing different lines of transgenic mice carrying various versions of a gene, therefore, permits cis acting DNA sequences involved in the specificity of expression to be defined, in the context of the developing animal. This in turn opens the way to a variety of experiments in which a given gene product is targeted to one or another cell type, thus offering some insight into the physiological role of this product. Such a strategy has been used, for example, to address the questions of the role of oncogenes in malignant transformation. The insertion of foreign DNA per se may disrupt the function of endogenous genes, thus creating an insertional mutation. The corresponding affected genes may subsequently be cloned, using the transgene as a tag. Finally, the ability to perform homologous recombination, recently demonstrated with embryonic stem cells that can colonize the germ line of a foreign embryo, should constitute in the near future a unique way to analyse in detail the functioning of the mammalian genome.Key words: transgenic mice, oncogenes, insertional mutagenesis, cis-acting sequences, homologous recombination.

scholarly journals All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

2009 ◽  
Vol 23 (12) ◽  
pp. 4335-4343 ◽  
Author(s):  
Eiichi Okamura ◽  
Hitomi Matsuzaki ◽  
Andrew D. Campbell ◽  
James Douglas Engel ◽  
Akiyoshi Fukamizu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Enhancer Activity

scholarly journals Yeast artificial chromosomes: an alternative approach to the molecular analysis of mouse developmental mutations

1990 ◽  
Vol 56 (2-3) ◽  
pp. 203-208 ◽  
Author(s):  
Zoia Larin ◽  
Hans Lehrach
Keyword(s):  
Mouse Genome ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Artificial Chromosome ◽  
Molecular Study ◽  
Insert Size ◽  
Artificial Chromosomes ◽  
Partial Digestion ◽  
Genomic Regions ◽  
Yeast Artificial Chromosomes

SummaryMammalian genetics now allows a molecular study of genomic regions previously analysed by genetic and embryological techniques. To simplify such an analysis, we have established a number of libraries of mouse DNA in Yeast Artificial Chromosome (YAC) vectors, constructed either by partial digestion with EcoRI, or by complete digestion with enzymes which cut rarely in the mammalian genome. In this paper we report the construction of complete digest libraries prepared from mouse genomic DNA using the rare cutter enzymes NoiI and BssHII, and the detection of gene loci from the H-2 complex, the t–complex, and other loci from the mouse genome. Due to their large insert size, YAC clones simplify the cloning of extended regions of the mouse genome surrounding known developmental mutations and should, after introduction into the germ line, offer a high probability of correct expression of the genes contained within the cloned region. We hope that this will allow the use of YAC clones to scan regions of interest such as the t–complex for specific genes by testing DNA introduced into transgenic mice for the ability to complement mutations localised to this region.

scholarly journals An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7682-7695 ◽  
Author(s):  
Tomohiro Tsuduki ◽  
Megumi Nakano ◽  
Nao Yasuoka ◽  
Saeko Yamazaki ◽  
Teruaki Okada ◽  
...  
Keyword(s):  
Yeast Artificial Chromosome ◽  
Fluorescent Protein ◽  
De Novo ◽  
Artificial Chromosome ◽  
Time Lapse ◽  
Living Cells ◽  
Lac Repressor ◽  
Host Chromosome ◽  
Artificial Chromosomes ◽  
Spindle Midzone

ABSTRACT Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing α-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input α-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

1987 ◽  
Vol 7 (11) ◽  
pp. 4024-4029
Author(s):  
M Trudel ◽  
J Magram ◽  
L Bruckner ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Base Pairs ◽  
Beta Globin Gene ◽  
Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice

Nature ◽  
1993 ◽  
Vol 362 (6417) ◽  
pp. 258-261 ◽  
Author(s):  
Andreas Schedl ◽  
Lluís Montoliu ◽  
Gavin Kelsey ◽  
Günther Schütz
Keyword(s):  
Transgenic Mice ◽  
Copy Number ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Tyrosinase Gene
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