Kit for constructing transposon and use thereof
Inventors
Assignees
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Publication Number
US-11299748-B2
Publication Date
2022-04-12
Expiration Date
Abstract
A kit for constructing a transposon is provided. The kit comprises five plasmids, each of which consists essentially of a gene cassette linked or not linked to a terminal repeat. The kit is useful in selecting the type of piggyBac that exhibits the least enhancer activity in the host cells.
Core Innovation
The invention provides a piggyBac terminal-repeat selection kit and a method of constructing a transposon for introducing an exogenous gene into host cells. A control plasmid includes a gene cassette having a promoter of SEQ ID NO: 5 and a reporter gene operably linked to the promoter, and first to fourth plasmids include the same gene cassette with terminal repeats disposed upstream of the gene cassette, where the first to fourth terminal repeats respectively comprise the nucleotide sequences of SEQ ID NOs: 1 to 4.
The method determines a control expression level of the reporter gene from the control plasmid and compares respective expression levels from the reporter genes carried by the first to fourth plasmids in the host cells. A transposon is then constructed that includes an expression cassette comprising a non-prokaryotic promoter and an exogenous gene operably linked to the non-prokaryotic promoter, together with a first selected terminal repeat and a second selected terminal repeat. The first selected terminal repeat is one of the first to fourth terminal repeats that elicits an expression level closest to the control expression level, and the second selected terminal repeat is selected in a complementary paired manner.
The transposon construction defines the disposition of the paired terminal repeats relative to the expression cassette and gene cassette based on which terminal repeat is selected first. When the first selected terminal repeat is the first or the third terminal repeat, it is disposed upstream of the expression cassette and the second selected terminal repeat is the second or the fourth terminal repeat disposed downstream of the gene cassette; when the first selected terminal repeat is the second or the fourth terminal repeat, it is disposed downstream of the expression cassette and the second selected terminal repeat is the first or the third terminal repeat disposed upstream of the gene cassette. The described kit includes the control plasmid and the first to fourth plasmids carrying the terminal repeat nucleotide sequences of SEQ ID NOs: 1 to 4.
Claims Coverage
The independent claims are clm-00001 and clm-00011. clm-00001 claims the method for selecting terminal repeats by reporter expression comparison and using the selected paired terminal repeats to construct a transposon with an expression cassette for introducing an exogenous gene into host cells. clm-00011 claims a kit containing a control plasmid and first-to-fourth plasmids each with terminal repeats disposed upstream of the gene cassette.
Terminal-repeat selection using control and test reporter expression levels
A method comprising transfecting the host cells with a control plasmid and first to fourth plasmids having respective terminal repeats disposed upstream of the gene cassette, determining a control expression level of the reporter gene and respective expression levels of the reporter genes, and constructing a transposon with a first selected terminal repeat that elicits an expression level closest to the control expression level.
Paired first and second selected terminal repeats with conditional upstream/downstream disposition
Constructing the transposon that consists essentially of an expression cassette comprising a non-prokaryotic promoter and an exogenous gene operably linked to the non-prokaryotic promoter, a first selected terminal repeat, and a second selected terminal repeat, with upstream/downstream disposition determined by whether the first selected terminal repeat is the first or third terminal repeat, or the second or fourth terminal repeat.
Control plasmid and test plasmids with SEQ ID NO: 1-4 terminal repeats in a kit
A kit comprising a control plasmid consisting essentially of a gene cassette comprising a promoter of SEQ ID NO: 5 and a reporter gene operably linked to the promoter, and first to fourth plasmids consisting essentially of the gene cassette and first to fourth terminal repeats disposed upstream of the gene cassette, wherein the first to fourth terminal repeats respectively consist of the nucleotide sequences of SEQ ID NOs: 1 to 4.
Across the independent claims, the core coverage is terminal-repeat choice by comparing reporter expression from a control plasmid versus plasmids carrying terminal repeat variants (SEQ ID NOs: 1-4), followed by constructing a transposon expression cassette that uses a first and a second selected terminal repeat selected as specified and arranged upstream/downstream based on which terminal repeat is chosen first. The kit claim covers the corresponding plasmid components that enable the selection.
Stated Advantages
microR/selected repeats minimize enhancer effects in specified host cells for safer exogenous gene expression and disease therapy applications.
Documented Applications
Disease treatment applications for cancer, inflammatory, immunodeficiency, angiogenesis-associated, and hormone-associated conditions.
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