Optimized host cells for protein production
First Claim
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1. A method for isolating a cell with an increased rate of cell proliferation, comprising the steps of:
- contacting a population of cells with a fluorescent reagent for monitoring the rate of cell proliferation; and
isolating the cell that exhibits a level of fluorescence of the fluorescent reagent that correlates with increased cell proliferation.
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Abstract
The present invention relates to methods for isolating cells that express increased levels of an RNA or protein of interest, wherein the cells exhibit altered growth profiles, such as cells with increased or decreased rates of proliferation, increased or decreased rates of apoptosis, or cells with a biphasic growth profile.
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Citations
51 Claims
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1. A method for isolating a cell with an increased rate of cell proliferation, comprising the steps of:
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contacting a population of cells with a fluorescent reagent for monitoring the rate of cell proliferation; and isolating the cell that exhibits a level of fluorescence of the fluorescent reagent that correlates with increased cell proliferation. - View Dependent Claims (2)
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3. A method for isolating a cell with an increased rate of cell proliferation, wherein the cell also expresses high levels of an RNA of interest, comprising the steps of:
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contacting a population of cells with a fluorogenic probe that fluoresces upon hybridization to said RNA of interest; contacting said population with a fluorescent reagent for monitoring the rate of cell proliferation, wherein the reagent fluoresces at a wavelength different than that of the fluorogenic probe; and isolating the cell that exhibits increased fluorescence of the fluorogenic probe and a level of fluorescence of the fluorescent reagent that correlates with increased cell proliferation. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of producing a cell culture with increased cell density, wherein cells in the cell culture express high levels of an RNA of interest, comprising the steps of:
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contacting a population of cells with a fluorogenic probe that fluoresces upon hybridization to said RNA of interest; isolating a cell from the population that exhibits increased fluorescence of the fluorogenic probe; culturing the isolated cell to produce a first cell culture; repeating the previous steps to isolate a second cell culture; comparing the maximum-attained density of the first and second cell cultures; and identifying the cell culture with increased cell density wherein cells in the cell culture expresses high levels of the RNA of interest. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for isolating a cell with a biphasic growth profile, wherein the cell has an increased rate of proliferation in a first portion of the growth profile, and wherein the cell has a decreased rate of proliferation in a second portion of the growth profile, comprising the steps of:
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contacting a first population of cells with a fluorescent reagent for monitoring the rate of cell proliferation; culturing said population of cells at low cell density, isolating a cell from said population of cells that exhibits altered fluorescence of the fluorescent reagent in the first portion of the growth profile; culturing the isolated cell to produce a second population of cells; contacting said second population of cells with a fluorescent reagent for monitoring the rate of cell proliferation; culturing said second population of cells at high cell density; and
,isolating a cell exhibiting an unaltered or slower rate of alteration of fluuorescence of the fluorescent reagent in the second portion of the growth profile as compared to the altered fluorescence of the fluorescenct reagent in the first portion of the growth profile, thereby isolating a cell with a biphasic growth profile, wherein the cell has an increased rate of proliferation in a first portion of the growth profile, and wherein the cell has a decreased rate of proliferation in a second portion of the growth profile.
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34. A method for isolating a cell with a biphasic growth profile, wherein the cell has an increased rate of proliferation in a first portion of the growth profile, and wherein the cell has a decreased rate of proliferation in a second portion of the growth profile, and wherein the cell expresses high levels of an RNA of interest in the second portion of the growth profile, comprising the steps of:
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contacting a population of cells with a fluorogenic probe that fluoresces upon hybridization to said RNA of interest; contacting said population with a fluorescent reagent for monitoring the rate of cell proliferation, wherein the reagent fluoresces at a wavelength different than that of the fluorogenic probe; and isolating a cell that exhibits altered fluorescence of the fluorescent reagent in the first portion of the growth profile, unaltered or decreased change of fluorescence of the fluorescent reagent in the second portion of the growth profile, and increased fluorescence of the fluorogenic probe in the second portion of the growth profile. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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Specification