Stable cell lines resistant to apoptosis and nutrient stress and methods of making same
AssigneeBoyce Thompson Institute for Plant Research, Inc.
InventorsGary Blissard, Robert R. Granados, Guangyun Lin
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Abstract
Cell lines that are commonly used for protein expression are engineered to include genes that encode suppressors of apoptosis (SA). Insect cell lines expressing these SA genes are resistant to apoptosis or programmed cell death, and express certain recombinant proteins at increased levels. These cell lines also have increased resistance to many types of stress. Because some of the SA proteins inhibit apoptosis in a wide spectrum of organisms, these genes may be inserted into other plant or animal cell lines for a variety of purposes involving resistance to apoptosis or resistance to stress.
Claims7
A composition comprising a baculovirus expression system for expressing a recombinant protein, including:
- a) a baculovirus that has a functional p35 gene, and is engineered to express a recombinant protein;
- b) a cultured Sf9 cell line that is susceptible to infection by said baculovirus, and is engineered by transfection with a first recombinant expression vector to express a recombinant polynucleotide that encodes AcNPV p35, wherein said recombinant polynucleotide is cloned into said first recombinant expression vector such that said recombinant polynucleotide is capable of being expressed in said cell line;
- c) said first recombinant expression vector being delivered into at least one cell of said cultured cell line;
- and
- d) said at least one cell being infected by said baculovirus;
- e) such that apoptosis is inhibited, when said engineered cell line is infected by said baculovirus that includes a functional p35 gene.
A method for expressing a recombinant protein in a baculovirus expression system, comprising the steps of:
- a) engineering a baculovirus that has a functional p35 gene to express a recombinant protein;
- b) culturing an Sf9 cell line that is susceptible to infection by said baculovirus, and is engineered by transfection with a first recombinant expression vector to express a recombinant polynucleotide that encodes AcNPV p35, wherein said recombinant polynucleotide is cloned into said first recombinant expression vector such that said recombinant polynucleotide is capable of being expressed in said cell line;
- c) delivering said first recombinant expression vector into at least one cell of said cultured cell line;
- and
- d) infecting said at least one cell with said baculovirus, such that apoptosis is inhibited, when said engineered cell line is infected by said baculovirus that includes a functional p35 gene.
A method of developing a cell line containing a suppressor of apoptosis, comprising the steps of:
- a) isolating a recombinant polynucleotide that encodes AcNPV p35;
- b) constructing a first recombinant expression vector wherein said recombinant polynucleotide is cloned into said first recombinant expression vector such that said recombinant polynucleotide is capable of being expressed in said cell line;
- c) delivering said first recombinant expression vector into at least one Sf9 cell;
- d) exposing said at least one cell to any inducer of apoptosis;
- and
- e) selecting said cell lines from said at least one cell which survives exposure to said inducer of apoptosis, such that apoptosis induced by a subsequent baculovirus infection is inhibited, when said baculovirus includes a functional p35 gene.
A cultured cell line comprising an Sf9 insect cell line that is susceptible to infection by a baculovirus, and is engineered to stably express a recombinant polynucleotide that encodes AcNPV p35.
A cell line selected from the group consisting of ATCC accession number PTA-3099 and ATCC accession number PTA-3100.
Dependencies are inferred from the claim text.