Stability of HPRT Marker Gene Expression at Different Gene-Targeted Loci: Observing and Overcoming a Position Effect

Melton, David W.; Ketchen, Ann-Marie; Selfridge, Jim

doi:10.1093/nar/25.19.3937

Cited by 16 publications

(2 citation statements)

References 31 publications

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“…These results indicate that the DNA has become highly de novo methylated in all ES clones lacking the enhancer and the Sp1 sequences. While promoter strength is probably a factor influencing de novo methylation, these data imply that cis-acting sequences can inhibit de novo methylation over several kb, as suggested elsewhere (51).…”

Section: Dna Methylation Patterns and Luciferase Expression Of The Svsupporting

confidence: 71%

Factors Affecting de Novo Methylation of Foreign DNA in Mouse Embryonic Stem Cells

Hertz¹,

Schell

Doerfler

1999

Journal of Biological Chemistry

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Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones.Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation.

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Section: Dna Methylation Patterns and Luciferase Expression Of The Svsupporting

confidence: 71%

Factors Affecting de Novo Methylation of Foreign DNA in Mouse Embryonic Stem Cells

Hertz¹,

Schell

Doerfler

1999

Journal of Biological Chemistry

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“…Future transgenic livestock will probably have the transgene integrated into a permissive site. Although the appropriate genomic site remains to be determined, the approach has been evaluated in the mouse model (Bronson et al, 1996;Melton et al, 1997).…”

Section: Nuclear Transfer: a New Era For Cell Culturementioning

confidence: 99%

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Whitelaw¹,

Farini²,

Webster³

1999

Cytotechnology

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Transgenesis may allow the generation of farm animals with altered phenotype, animal models for research and animal bioreactors. Although such animals have been produced, the time and expense involved in generating transgenic livestock and then evaluating the transgene expression pattern is very restrictive. If questions about the ability and efficiency of expression could be asked solely in vitro rapid progress could be achieved. Unfortunately, experiments addressing transcriptional control in vitro have proved unreliable in their ability to indicate whether a transgene will be transcribed or not. However, initial studies suggest that cell culture may be able to predict in vivo post-transcriptional events. We review these issues and propose that strategies which engineer the transgene integration site could enhance the probability for efficient expression. This approach has now become feasible with the development of techniques allowing animals to be generated from somatic cells by nuclear transfer. The important step in this procedure is the use of cells grown in culture as the source of genetic information, allowing the selection of specific transgene integration events. This technology which has dramatically increased the potential use of transgenic livestock for both agricultural and biotechnological applications, is based on standard cell culture methodology. We are now at the start of a new era in large animal transgenics.

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Minigene

Wilson¹

2002

Encyclopedia of Molecular Biology

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Stability of HPRT Marker Gene Expression at Different Gene-Targeted Loci: Observing and Overcoming a Position Effect

Cited by 16 publications

References 31 publications

Factors Affecting de Novo Methylation of Foreign DNA in Mouse Embryonic Stem Cells

Factors Affecting de Novo Methylation of Foreign DNA in Mouse Embryonic Stem Cells

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