Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

Gnirke, Andreas; Huxley, Clare; Peterson, K.; Olson, Maynard V.

doi:10.1006/geno.1993.1121

Cited by 91 publications

(65 citation statements)

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“…One is the purification and concentration of the YAC, intact and in microinjectable form, and the other is the detailed analysis of the integrated fragments in the transgenic mice obtained. The general logic of isolating YACs from agarose via agarase treatment and the protection from random shearing by compaction of the DNA, as we have followed here, has been described in detail by Gnirke et al (1993) and . There are differences, however, in the methods followed for concentrating and purifying the YAC DNA into microinjection buffer.…”

Section: Discussionmentioning

confidence: 99%

A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection

et al. 1996

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Background: In recent years, interest has been revived in the possibility of transplanting organs into humans from a phylogenetically disparate species such as the pig (xenotransplantation). Such discordant xenografts, however, are subject to hyperacute rejection (HAR) and activation of host complement plays a major role in this rejection. This problem may be solved through the use of transgenic technology by providing the grafted tissue with molecules that down-regulate the action of host complement.

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Section: Discussionmentioning

confidence: 99%

A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection

et al. 1996

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“…The AHS3 j3-YAC was purified and microinjected into fertilized mouse eggs as described previously (26)(27)(28)(29) …”

Section: Methodsmentioning

confidence: 99%

“…pRS406 AHS3 was linearized with SphI (GenBank coordinate 7327), which cuts asymmetrically relative to the deletion breakpoints and gives recombination intervals of 1532 and 437 bp. Selection and identification of yeast isolates containing correct YIP insertions into the 13-YAC and selection and structural confirmation of proper YIP excisions were performed as described previously (27).The AHS3 j3-YAC was purified and microinjected into fertilized mouse eggs as described previously (26)(27)(28)(29) …”

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confidence: 99%

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

Peterson

Clegg

Navas

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

108

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To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on 0-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a p-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of e-globin gene expression and an increase of y-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of £-, y-, and f3-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.The human ,3-globin locus spans approximately 82 kb of chromosome 11. The locus consists of a powerful upstream control element, the locus control region (LCR), and five functional 13-like globin genes arrayed 5' to 3' in the order in which they are expressed during development. Six DNase I hypersensitive sites (HSs) flank the globin genes (1, 2). One site is located approximately 20 kb downstream from the ,B-globin gene (3'HS1), and five sites are located 6-22 kb upstream of the e-globin gene (5'HS1 to 5'HS5). 5'HS5 is constitutive, while 5'HS1-4 are erythroid-specific (1-5). The LCR activates the 13-globin locus chromosomal domain, insulates the globin genes from the effects of surrounding chromatin, restricts globin gene expression to cells of the erythroid lineage, and acts as a powerful enhancer directing high levels of globin production in erythroid cells (refs. 4 and 6; for review, see refs. 7 and 8).Most functional analyses of the DNase I hypersensitive sites of the LCR have been carried out in transgenic mice using simple constructs linking an individual HS region to a single human globin gene or to the G.y-3 genes in a cosmid (9-25).However, these constructs only test the ability of the individual sites to function as enhancers of linked transgene expression, and they do not reveal their role in the context of the native LCR. Testing the contribution of each HS to LCR function requires analysis of the effects of individual HS mutations in the context of the whole 1-globin locus. Synthesizing constructs of the 82-kb locus containing these mutations is technically difficult. We previously reported that the analysis of the regulation of the 13-like globin genes is greatly facilitated using ,3-globin locus yeast artificial chromosomes (13-YACs) (26,27).In this paper, we examine the effects of deletion of 5'HS3 or 5'HS2 of the 13-globin locus YAC on human 13-like globin gene expression. We demonstrate that when 5'HS3 is deleted, e-globin gene expression is decreased in the embryonic yolk sac, while deletion of 5'HS2 results in a min...

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“…PCR products were gel purified and sequenced using standard fluorescent dye terminator chemistry (Perkin Elmer, Norwalk, CT). Pulsed-field gel-purified YAC DNA was isolated as described (Gnirke et al 1993) and cleaned up by phenol extraction and ethanol precipitation. Random sequences from purified YAC DNA for STS development were obtained by constructing complete EcoRI, SacI, and EcoRI-SacI digest libraries in pBluescript SK (Stratagene, La Jolla, CA) or RsaI and HaeIII digest libraries in pSP72 (Promega, Madison, WI), and sequencing the insert ends of randomly picked plasmid clones.…”

Section: Methods Yacsmentioning

confidence: 99%

Clone–Contig and STS Maps of the Hereditary Hemochromatosis Region on Human Chromosome 6p21.3–p22

Lauer¹,

Meyer²,

Prass³

et al. 1997

Genome Res.

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YAC-based and bacterial-clone based STS-content maps were constructed that served as the framework physical maps for the positional cloning of a candidate gene for hereditary hemochromatosis. The YAC-based map comprises 43 YACs and 86 STS and spans ∼8 Mb of DNA between the class I region of the major histocompatibility complex on human chromosome 6p21.3 and D6S276 in 6p22. Comparison with published maps revealed a hole in the MIT/Whitehead and CEPH YAC maps that includes the immediate region around the hemochromatosis gene itself. Approximately 3 Mb of DNA was covered by a bacterial clone contig that consists of 38 BACs, 45 PACs, 26 P1 clones and one λ phage. The bacterial clone-based STS map comprises 153 STSs. A contiguous block of 8 STSs could be amplified from both human chromosome 6 and 5. Further characterization of selected STSs and bacterial clones by radiation hybrid mapping and fluorescence in situ hybridization, respectively, revealed the presence of a multicopy DNA segment, more than one bacterial clone length in size, which is duplicated near the chromosome-6 centromere and part of which is present in multiple copies on chromosome 5. Possible implications of the incomplete public YAC–contig map and of the multicopy segment for physical mapping and linkage disequilibrium studies of the hemochromatosis candidate region are discussed.[The sequence data described in this paper have been submitted to GenBank under accession nos. G31205–G31325.]

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Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

Cited by 91 publications

References 0 publications

A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection

A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

Clone–Contig and STS Maps of the Hereditary Hemochromatosis Region on Human Chromosome 6p21.3–p22

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