Monitoring of cells and trans-activating transcription elements

Claim 1

A method for studying a regulatory element of a transcriptional initiation regulatory system in a viable mammalian cell, said cell comprising a non-wild type expression construct comprising a transcriptional initiation regulatory region comprising a regulatory sequence of interest and a structural gene encoding β-galactosidase, wherein said β-galactosidase employs as a substrate a β-galactosidyl ether of a fluorescent phenolic compound that produces a fluorescent product upon hydrolysis of said ether, said method comprising: substantially irreversibly introducing said substrate into said cell in a hypotonic solution at a temperature in the range of about 25°-40° C.; incubating said cell at a temperature below about the membrane freezing point; and detecting said fluorescent product in said cell as a measure of the activity of said regulatory element.

Claim 2

A method for studying a regulatory element of a transcriptional initiation regulatory system in a viable mammalian cell, said cell comprising a non-wild type expression construct comprising a transcriptional initiation regulatory region comprising a regulatory sequence of interest and a structural gene encoding β-galactosidase, wherein said β-galactosidase employs as a substrate a β-galactosidyl ether of a fluorescent phenolic compound that produces a fluorescent product upon hydrolysis of said ether, said method comprising: substantially irreversibly introducing said substrate into said cell in a hypotonic solution at a temperature in the range of about 25°-40° C.; incubating said cell at a temperature below about the membrane freezing point; and detecting said fluorescent product in said cell as a measure of the activity of said regulatory element.

Claim 7

A method for producing a fluorescent product in a viable mammalian cell, said cell comprising a non-wild type expression construct encoding the enzyme β-galactosidase, said method comprising: substantially irreversibly introducing under a first set of conditions fluorescein di-β-galactopyranoside or derivative thereof as the enzyme substrate into said cell, wherein said substrate is characterized by being capable of substantially irreversible introduction into said viable cell and produces a product which is substantially retained in said cell under the conditions of producing said fluorescent product; incubating said cell under a second set of conditions at a lower temperature than said first set of conditions, where said fluorescent product is produced; wherein said first set of conditions comprises a hypotonic solution and a temperature in the range of about 25°-40° C. and said second set of conditions comprises a temperature below about the membrane freezing point; and detecting said fluorescent product in said cell.

Claim 8

A method for producing a fluorescent product in a viable mammalian cell, said cell comprising a non-wild type expression construct encoding the enzyme β-galactosidase, said method comprising: substantially irreversibly introducing under a first set of conditions fluorescein di-β-galactopyranoside or derivative thereof as the enzyme substrate into said cell, wherein said substrate is characterized by being capable of substantially irreversible introduction into said viable cell and produces a product which is substantially retained in said cell under the conditions of producing said fluorescent product; incubating said cell under a second set of conditions at a lower temperature than said first set of conditions, where said fluorescent product is produced; wherein said first set of conditions comprises a hypotonic solution and a temperature in the range of about 25°-40° C. and said second set of conditions comprises a temperature below about the membrane freezing point; and detecting said fluorescent product in said cell.

Claim 10

A method for detecting the presence of an infectious virus in a sample, said method employing a test viable cell susceptible to said virus and comprising a non-wild type expression construct comprising a transcriptional initiation regulatory region including a regulatory sequence which is responsive to a trans acting entity produced by said virus and a structural gene encoding β-galactosidase under the transcriptional initiation regulation of said regulatory region, said method comprising: cultivating said test viable cell with said sample under conditions wherein said test viable cell becomes infected with any infectious virus present in said sample; substantially irreversibly introducing into said cell as the enzyme substrate fluorescein di-β-galactopyranoside or derivative thereof by means of a hypotonic solution of said substrate at a temperature in the range of about 25°-40° C. for a time sufficient to introduce an excess of substrate and minimize reaction of said substrate and then cooling said cell to a temperature below about the membrane freezing point; incubating said cell for a sufficient time for said fluorescent product to form; and detecting said fluorescent product as a measure of the presence of said infectious virus in said sample.

Claim 11

A method for detecting the presence of an infectious virus in a sample, said method employing a test viable cell susceptible to said virus and comprising a non-wild type expression construct comprising a transcriptional initiation regulatory region including a regulatory sequence which is responsive to a trans acting entity produced by said virus and a structural gene encoding β-galactosidase under the transcriptional initiation regulation of said regulatory region, said method comprising: cultivating said test viable cell with said sample under conditions wherein said test viable cell becomes infected with any infectious virus present in said sample; substantially irreversibly introducing into said cell as the enzyme substrate fluorescein di-β-galactopyranoside or derivative thereof by means of a hypotonic solution of said substrate at a temperature in the range of about 25°-40° C. for a time sufficient to introduce an excess of substrate and minimize reaction of said substrate and then cooling said cell to a temperature below about the membrane freezing point; incubating said cell for a sufficient time for said fluorescent product to form; and detecting said fluorescent product as a measure of the presence of said infectious virus in said sample.