Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 ( Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1 b-m3 locus allowing allele specific PCR assays...

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Bibliographic Details
Published in:Genomics (San Diego, Calif.) Calif.), 2009-03, Vol.93 (3), p.196-204
Main Authors: Yang, G.S., Banks, K.G., Bonaguro, R.J., Wilson, G., Dreolini, L., de Leeuw, C.N., Liu, L., Swanson, D.J., Goldowitz, D., Holt, R.A., Simpson, E.M.
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Cytomegalovirus - genetics
Diverse techniques
Embryonic stem cells
Embryonic Stem Cells - cytology
Embryonic Stem Cells - metabolism
Female
Fundamental and applied biological sciences. Psychology
Gene Expression Profiling - methods
Gene Knock-In Techniques - methods
Gene targeting
Genes. Genome
Genetic Vectors - genetics
Genetics of eukaryotes. Biological and molecular evolution
Genomics
Genomics - methods
Hprt1
Human cytomegalovirus
Humans
Hypoxanthine Phosphoribosyltransferase - genetics
Knock-in
Male
Mice
Mice, Transgenic
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Promoter regions
Promoter Regions, Genetic - genetics
Reproducibility of Results
Sequence Alignment
Sequence Deletion
Transgenic
Vectors
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Description
Summary:We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 ( Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1 b-m3 locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project ( http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.
ISSN:0888-7543
1089-8646
DOI:10.1016/j.ygeno.2008.09.014