US5272071A
Expired / Lapsed
US
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Method for the modification of the expression characteristics of an endogenous gene of a given cell line
Assignee: Merck Serono SA
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Filing: 1990-12-21
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Granted: 1993-12-21
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Expires: 2010-12-21
Abstract
Abstract Normally transcriptionally silent genes in a cell line or microorganism may be activated for expression by inserting a DNA regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell or which is promiscuous, the regulatory element being inserted so as to be operatively linked with the normally silent gene in question. The insertion is accomplished by means of homologous recombination by creating a DNA construct including a segment having a DNA segment of the normally silent gene (targeting DNA) and the DNA regulatory element to induce gene transcription. The technique is also used to modify the expression characteristics of any endogenous gene of a given cell line or microorganism.
Inventors
- 👤 Scott C. Chappel
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 03:25
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/67; C12N15/87; C12N15/90; C12N15/902; C12N15/907
Claims Analysis
Total Claims: 66
Claim 1
Independent Claim
98 words
A method of activating a predetermined normally transcriptionally silent gene within the genome of a cell line so as to enable said cell line to express the gene product of said gene, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising a DNA regulatory segment capable of stimulating expression of said gene when operatively linked thereto and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said regulatory segment is operatively linked to said gene of interest.
Claim 2
Independent Claim
98 words
A method of activating a predetermined normally transcriptionally silent gene within the genome of a cell line so as to enable said cell line to express the gene product of said gene, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising a DNA regulatory segment capable of stimulating expression of said gene when operatively linked thereto and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said regulatory segment is operatively linked to said gene of interest.
Claim 3
Independent Claim
94 words
A method of modifying, the expression characteristics of predetermined a gene within the genome of a cell line, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising a DNA regulatory segment capable of modifying the expression characteristics of said gene when operatively linked thereto, as compared to its existing DNA regulatory segment, and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said regulatory segment is operatively linked to said gene of interest.
Claim 4
Independent Claim
94 words
A method of modifying, the expression characteristics of predetermined a gene within the genome of a cell line, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising a DNA regulatory segment capable of modifying the expression characteristics of said gene when operatively linked thereto, as compared to its existing DNA regulatory segment, and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said regulatory segment is operatively linked to said gene of interest.
Claim 5
Independent Claim
97 words
A method of modifying the expression characteristics of a predetermined gene within the genome of a cell line, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising an expressible, amplifiable gene capable of amplifying said gene when inserted in sufficiently close proximity thereto, and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said amplifiable gene is in sufficiently close proximity to said gene of interest to cause amplification thereof when said amplifiable gene is amplified.
Claim 6
Independent Claim
97 words
A method of modifying the expression characteristics of a predetermined gene within the genome of a cell line, comprising inserting a DNA construct into said genome by homologous recombination, said DNA construct comprising an expressible, amplifiable gene capable of amplifying said gene when inserted in sufficiently close proximity thereto, and a DNA targeting segment homologous to a region of said genome within or proximal to said gene, wherein said construct is inserted such that said amplifiable gene is in sufficiently close proximity to said gene of interest to cause amplification thereof when said amplifiable gene is amplified.
Claim 7
Dependent Claim
31 words
A method in accordance with claim 3, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said expressible, amplifiable gene.
Claim 8
Dependent Claim
55 words
A method in accordance with claim 1, 2 wherein said DNA construct comprises two DNA targeting segments, each homologous to a region of said genome within or proximate to said gene, one of said targeting segments being upstream of said regulatory segment and the other of said targeting segments being downstream of said regulatory segment.
Claim 9
Dependent Claim
30 words
A method in accordance with claim 1, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said regulatory segment.
Claim 10
Dependent Claim
58 words
A method in accordance with claim 1, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted.
Claim 11
Dependent Claim
27 words
A method in accordance with claim 1, wherein said DNA construct additionally comprises an expressible amplifiable gene disposed so as to be inserted with said regulatory segment.
Claim 12
Dependent Claim
16 words
A method in accordance with claim 1, wherein said cell line is a eukaryotic cell line.
Claim 13
Dependent Claim
16 words
A method in accordance with claim 9, wherein said cell line is an animal cell line.
Claim 14
Dependent Claim
16 words
A method in accordance with claim 9, wherein said cell line is a mammalian cell line.
Claim 15
Dependent Claim
16 words
A method in accordance with claim 9, wherein said cell line is a plant cell line.
Claim 16
Dependent Claim
59 words
A method in accordance with claim 6, and additionally for causing expression of said gene product, further including the steps of, following said inserting step: selecting clones of said cell line which express the product of said selectable marker gene; cultivating the selected clones under conditions sufficient to permit expression of said gene product; and collecting said gene product.
Claim 17
Dependent Claim
28 words
A method in accordance with claim 13, wherein said selectable marker gene is the neomycin resistance gene and said selecting step comprises selecting those clones having neomycin resistance.
Claim 18
Dependent Claim
76 words
A method in accordance with claim 13 wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted, and said selecting step further includes selecting those clones which do not express said negative selectable marker gene.
Claim 19
Dependent Claim
42 words
A method in accordance with claim 15 wherein said negative selectable marker gene is the Herpes Simplex Virus thymidine kinase gene and said selecting step includes selecting those clones which survive exposure to a medium that kills cells which express said gene.
Claim 20
Dependent Claim
45 words
A genome of a cell line, the genome having a DNA regulatory segment operatively linked with a predetermined naturally occurring gene at an insertion site characterized by a predetermined DNA sequence, said DNA regulatory segment not being naturally occurring at said location in the genome.
Claim 21
Dependent Claim
45 words
A genome of a cell line, the genome having a DNA regulatory segment operatively linked with a predetermined naturally occurring gene at an insertion site characterized by a predetermined DNA sequence, said DNA regulatory segment not being naturally occurring at said location in the genome.
Claim 22
Dependent Claim
58 words
A cell line capable of expressing a gene product by a predetermined normally transcriptionally silent gene within the genome of said cell line, said genome having inserted therein a DNA regulatory segment operatively linked with said normally transcriptionally silent gene, said DNA regulatory segment being capable of promoting the expression of a gene product by said cell line.
Claim 23
Dependent Claim
58 words
A cell line capable of expressing a gene product by a predetermined normally transcriptionally silent gene within the genome of said cell line, said genome having inserted therein a DNA regulatory segment operatively linked with said normally transcriptionally silent gene, said DNA regulatory segment being capable of promoting the expression of a gene product by said cell line.
Claim 24
Dependent Claim
75 words
A cell line capable of enhanced expression of a gene product compared to the cell line from which it is derived, said gene product being the expression product of predetermined an endogenous gene within the genome of said cell, said genome having inserted therein in an operative manner, at or near said endogenous gene, an exogenous DNA regulatory segment and/or amplifiable gene capable of enhancing the expression of said gene product by said cell line.
Claim 25
Dependent Claim
75 words
A cell line capable of enhanced expression of a gene product compared to the cell line from which it is derived, said gene product being the expression product of predetermined an endogenous gene within the genome of said cell, said genome having inserted therein in an operative manner, at or near said endogenous gene, an exogenous DNA regulatory segment and/or amplifiable gene capable of enhancing the expression of said gene product by said cell line.
Claim 26
Dependent Claim
23 words
A cell line in accordance with claim 19, wherein said exogenous DNA regulatory segment and/or amplifiable gene is an exogenous DNA regulatory segment.
Claim 27
Dependent Claim
22 words
A cell line in accordance with claim 19, wherein said exogenous DNA regulatory segment and/or amplifiable gene is an exogenous amplifiable gene.
Claim 28
Dependent Claim
32 words
A cell line in accordance with claim 18 wherein said DNA regulatory segment is one which is capable of promoting the expression of a gene product normally expressed by said cell line.
Claim 29
Dependent Claim
32 words
A cell line in accordance with claim 22, wherein the inserted DNA regulatory segment is part of a DNA construct comprising said DNA regulatory segment and at least one selectable marker gene.
Claim 30
Dependent Claim
17 words
A cell line in accordance with claim 23, wherein said DNA construct additionally comprises an amplifiable gene.
Claim 31
Dependent Claim
37 words
A method of obtaining a gene product from a cell line, comprising culturing a differentiated a cell line in accordance with claim 18 under conditions which permit expression of said gene product, and collecting said gene product.
Claim 32
Dependent Claim
54 words
A DNA construct for insertion into a predetermined host cell line, comprising a DNA regulatory segment capable of modifying the expression characteristics of genes in the host cell line when operatively linked thereto and a DNA targeting segment homologous to a region of the genome of a preselected gene within the host cell line.
Claim 33
Dependent Claim
54 words
A DNA construct for insertion into a predetermined host cell line, comprising a DNA regulatory segment capable of modifying the expression characteristics of genes in the host cell line when operatively linked thereto and a DNA targeting segment homologous to a region of the genome of a preselected gene within the host cell line.
Claim 34
Dependent Claim
54 words
A DNA construct for insertion into a predetermined host cell line, comprising an expressible, amplifiable gene capable of amplifying a gene in the host cell line when inserted in sufficiently close proximity thereto, and a DNA targeting segment homologous to a region of the genome of a preselected gene within the host cell line.
Claim 35
Dependent Claim
54 words
A DNA construct for insertion into a predetermined host cell line, comprising an expressible, amplifiable gene capable of amplifying a gene in the host cell line when inserted in sufficiently close proximity thereto, and a DNA targeting segment homologous to a region of the genome of a preselected gene within the host cell line.
Claim 36
Dependent Claim
54 words
A method in accordance with claim 2, wherein said DNA construct comprises two DNA targeting segments, each homologous to a region of said genome within or proximate to said gene, one of said targeting segments being upstream of said regulatory segment and the other of said targeting segments being downstream of said regulatory segment.
Claim 37
Dependent Claim
30 words
A method in accordance with claim 2, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said regulatory segment.
Claim 38
Dependent Claim
58 words
A method in accordance with claim 2, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted.
Claim 39
Dependent Claim
27 words
A method in accordance with claim 2, wherein said DNA construct additionally comprises an expressible amplifiable gene disposed so as to be inserted with said regulatory segment.
Claim 40
Dependent Claim
16 words
A method in accordance with claim 2, wherein said cell line is a eukaryotic cell line.
Claim 41
Dependent Claim
16 words
A method in accordance with claim 32, wherein said cell line is an animal cell line.
Claim 42
Dependent Claim
16 words
A method in accordance with claim 32, wherein said cell line is a mammalian cell line.
Claim 43
Dependent Claim
16 words
A method in accordance with claim 32, wherein said cell line is a plant cell line.
Claim 44
Dependent Claim
59 words
A method in accordance with claim 29, and additionally for causing expression of said gene product, further including the steps of, following said inserting step: selecting clones of said cell line which express the product of said selectable marker gene; cultivating the selected clones under conditions sufficient to permit expression of said gene product; and collecting said gene product.
Claim 45
Dependent Claim
28 words
A method in accordance with claim 36, wherein said selectable marker gene is the neomycin resistance gene and said selecting step comprises selecting those clones having neomycin resistance.
Claim 46
Dependent Claim
76 words
A method in accordance with claim 36, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted, and said selecting step further includes selecting those clones which do not express said negative selectable marker gene.
Claim 47
Dependent Claim
42 words
A method in accordance with claim 38, wherein said negative selectable marker gene is the Herpes Simplex Virus thymidine kinase gene and said selecting step includes selecting those clones which survive exposure to a medium that kills cells which express said gene.
Claim 48
Dependent Claim
54 words
A method in accordance with claim 3, wherein said DNA construct comprises two DNA targeting segments, each homologous to a region of said genome within or proximate to said gene, one of said targeting segments being upstream of said regulatory segment and the other of said targeting segments being downstream of said regulatory segment.
Claim 49
Dependent Claim
30 words
A method in accordance with claim 3, wherein said DNA construct additionally comprises at least one expressible selectable marker gene disposed so as to be inserted with said regulatory segment.
Claim 50
Dependent Claim
58 words
A method in accordance with claim 3, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment to as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted.
Claim 51
Dependent Claim
27 words
A method in accordance with claim 3, wherein said DNA construct additionally comprises an expressible amplifiable gene disposed so as to be inserted with said regulatory segment.
Claim 52
Dependent Claim
16 words
A method in accordance with claim 3, wherein said cell line is a eukaryotic cell line.
Claim 53
Dependent Claim
16 words
A method in accordance with claim 44, wherein said cell line is an animal cell line.
Claim 54
Dependent Claim
16 words
A method in accordance with claim 44, wherein said cell line is a mammalian cell line.
Claim 55
Dependent Claim
16 words
A method in accordance with claim 44, wherein said cell line is a plant cell line.
Claim 56
Dependent Claim
59 words
A method in accordance with claim 41, and additionally for causing expression of said gene product, further including the steps of, following said inserting step: selecting clones of said cell line which express the product of said selectable marker gene; cultivating the selected clones under conditions sufficient to permit expression of said gene product; and collecting said gene product.
Claim 57
Dependent Claim
28 words
A method in accordance with claim 48, wherein said selectable marker gene is the neomycin resistance gene and said selecting step comprises selecting those clones having neomycin resistance.
Claim 58
Dependent Claim
76 words
A method in accordance with claim 48, wherein said DNA construct additionally comprises a negative selectable marker gene disposed with respect to said targeting segment so as not to be inserted when said construct is properly inserted by homologous recombination, whereby said negative selectable marker is not expressed in cells in which said DNA construct is properly inserted, and said selecting step further includes selecting those clones which do not express said negative selectable marker gene.
Claim 59
Dependent Claim
42 words
A method in accordance with claim 50, wherein said negative selectable marker gene is the Herpes Simplex Virus thymidine kinase gene and said selecting step includes selecting those clones which survive exposure to a medium that kills cells which express said gene.
Claim 60
Dependent Claim
32 words
A cell line in accordance with claim 20, wherein said DNA regulatory segment is one which is capable of promoting the expression of a gene product normally expressed by said cell line.
Claim 61
Dependent Claim
35 words
A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 19 under conditions which permit expression of said gene product, and collecting said gene product.
Claim 62
Dependent Claim
35 words
A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 20 under conditions which permit expression of said gene product, and collecting said gene product.
Claim 63
Dependent Claim
35 words
A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 21 under conditions which permit expression of said gene product, and collecting said gene product.
Claim 64
Dependent Claim
35 words
A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 22 under conditions which permit expression of said gene product, and collecting said gene product.
Claim 65
Dependent Claim
35 words
A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 23 under conditions which permit expression of said gene product, and collecting said gene product.
Claim 66
Dependent Claim
35 words
A method of obtaining a gene product from a cell line, comprising culturing a cell line in accordance with claim 24 under conditions which permit expression of said gene product, and collecting said gene product.
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