Abstract
A method to assemble linear expression elements for rapid gene expression is described. Primers containing target specific sequences and N.Bpu10 I nickase recognition sites were used to amplify promoter, open reading frame and terminator fragments. Amplified fragments were treated with N.Bpu10 I nickase and exonuclease III to generate overhangs for directional ligation. These fragments were ligated and further amplified with element-specific primers. The amplified DNA was transfected into mammalian cells for gene expression.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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CHO Cells
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Cattle
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Cricetinae
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Cricetulus
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DNA / chemistry
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DNA / metabolism
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DNA Fragmentation / genetics*
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DNA Primers / chemistry
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Deoxyribonuclease I / chemistry*
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Deoxyribonucleases, Type II Site-Specific / chemistry*
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Deoxyribonucleases, Type II Site-Specific / metabolism
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Exodeoxyribonucleases / chemistry*
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Feasibility Studies
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Gene Expression Regulation / genetics*
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Growth Hormone / biosynthesis*
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Growth Hormone / genetics
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Polymerase Chain Reaction / methods
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Promoter Regions, Genetic / genetics
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Protein Engineering / methods*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / genetics
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Terminator Regions, Genetic
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Transfection / methods*
Substances
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DNA Primers
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Recombinant Proteins
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Growth Hormone
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DNA
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Exodeoxyribonucleases
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exodeoxyribonuclease III
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endodeoxyribonuclease Bpu10I
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Deoxyribonuclease I
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Deoxyribonucleases, Type II Site-Specific