Cytosolic forms for β-lactamase and uses thereof
AssigneeThe Regents of the University of California
InventorsRoger Y. Tsien, Gregor Zlokarnik
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Abstract
The present invention is directed to nucleic acid molecules that encode a cytosolic form of beta-lactamase, cells that include such nucleic acid molecules and methods for their use. These cells can be used in a variety of methods, such as methods for monitoring the expression of a gene or biochemical activity.
Claims35
A recombinant nucleic acid molecule comprising expression control sequences adapted for function in a vertebrate cell and operably linked to a nucleotide sequence coding for the expression of a β-lactamase, wherein the nucleic acid molecule further comprises preferred mammalian Kozak sequences for the β-lactamase and the nucleotide sequence coding for the expression of the β-lactamase lacks a signal sequence.
An expression vector comprising a recombinant nucleic acid molecule, comprising:
- expression control sequences adapted for function in an eukaryotic cell and operably linked to a nucleotide sequence coding for the expression of a cytosolic β-lactamase, wherein the nucleic acid molecule further comprises preferred mammalian Kozak sequences for the β-lactamase.
A mammalian host cell, transfected with a recombinant nucleic acid molecule further comprising expression control sequences adapted for function in a mammalian cell and operably linked to a nucleotide sequence coding for the expression of a β-lactamase,
- wherein the nucleic acid molecule further comprises mammalian Kozak sequences for the β-lactamase and the nucleotide sequence coding for the expression of the β-lactamase lacks a signal sequence.
A method for cellular clonal selection comprising,
- a) transfecting a host cell with a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising expression control sequences adapted for function in a vertebrate cell and operably linked to a nucleotide sequence coding for the expression of a β-lactamase, wherein the nucleic acid molecule further comprises preferred mammalian Kozak sequences for the β-lactamase, and the nucleotide sequence coding for the β-lactamase lacks a signal sequence,
- b) contacting said host cell with a membrane permeable substrate of β-lactamase, and
- c) selecting the cells based on fluorescence derived from the membrane permeable substrate of β-lactamase using a fluorescence activated cell sorter.
Dependencies are inferred from the claim text.