CELL LINES WITH MULTIPLE DOCKS FOR GENE INSERTION
The present invention relates to host cell lines containing multiple dock sites for insertion of a nucleic acid construct, and in particular nucleic acid constructs that express an exogenous gene of interest.
1 . A host cell comprising a genome, the genome comprising from 1 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element.
2 . The host cell of claim 1 , wherein the genome comprises from 5 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element.
3 - 5 . (canceled)
6 . The host cell of claim 1 , wherein the integrated docking sites are independently positioned throughout the genome.
7 . The host cell of claim 1 , wherein the dock site insertion element is targeted by enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.
8 . The host cell of claim 1 , wherein the dock site insertion element is selected from the group consisting of a recombinase dock site insertion element and a HDR dock site insertion element.
9 . The host cell of claim 8 , wherein the dock site insertion element is a recombinase dock site insertion element.
10 . The host cell of claim 9 , wherein the recombinase dock site insertion element comprises an attachment site (att).
11 . The host cell of claim 10 , wherein the attachment site (att) is selected from the group consisting of attB and attP and attR and attL.
12 . The host cell of claim 9 , wherein the recombinase dock site insertion element comprises a LoxP sequence.
13 . The host cell line of claim 9 , wherein the recombinase dock site insertion element is a Flp Recombination Target (FRT) site.
14 - 18 . (canceled)
19 . The host cell line of claim 1 , wherein each docking site is flanked by exogenous integrating vector sequences.
20 . The host cell line of claim 19 , wherein the exogenous integrating vector sequences are selected from the group consisting of viral vector sequences and transposon vector sequences.
21 . The host cell line of claim 1 , wherein the docking sites each further comprise a sequence encoding a selectable maker operably linked to a promoter.
22 . The host cell line of claim 1 , wherein the host cell line further comprises an exogenous sequence encoding an enzyme operably linked to a promoter.
23 . The host cell line of claim 1 , wherein the exogenous sequence encoding an enzyme operably linked to a promoter is inserted into the genome of the host cell.
24 . The host cell line of claim 23 , wherein the exogenous sequence encoding an enzyme operably linked to a promoter is inserted at the dock site.
25 . The host cell line of claim 1 , wherein the exogenous sequence encoding an enzyme operably linked to a promoter is provided in an episomal expression vector.
26 . (canceled)
27 . The host cell line of claim 25 , wherein the exogenous enzyme is selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.
28 - 29 . (canceled)
30 . The host cell line of claim 1 , wherein the dock site insertion element is positioned to facilitate cassette exchange.
31 . The host cell line of claim 1 , wherein each docking site comprises two dock site insertion elements.
32 . The host cell line of claim 31 , wherein the two dock site insertion elements are positioned to facilitate cassette exchange.
33 . (canceled)
34 . The host cell line of claim 1 , further comprising nucleic acid expression constructs inserted at 1 or more of the docking sites.
35 - 36 . (canceled)
37 . The host cell line of claim 34 , wherein the nucleic acid expression construct further comprises the following elements in operable association in 5′ to 3′ order:
a first promoter sequence;
a selectable marker sequence;
a second promoter sequence;
a nucleic acid sequence encoding a first protein of interest that is operably linked to the internal promoter; and
a poly A signal sequence.
38 . The host cell line of claim 37 , wherein the nucleic acid construct further comprising at least one insertion element at a position or positions selected from the group consisting of 5′ to the first promoter, 3′ to the poly A signal sequence, between the first promoter and the poly A signal sequence, between the selectable marker and the second promoter sequence, and both 5′ to the first promoter and 3′ to the poly A signal sequence.
39 - 105 . (canceled)
106 . A cell culture comprising host cells of claim 1 .
107 . (canceled)
108 . A process for producing a protein of interest comprising culturing host cells according to claim 34 conditions that the protein of interest is expressed and purifying the protein of interest from the host cell culture.
109 - 112 . (canceled)
113 . A system comprising:
a host cell of claim 1 ;
one or more nucleic acid expression constructs, wherein the nucleic acid expression construct further comprises the following elements in operable association in 5′ to 3′ order:
a selectable marker sequence;
an internal promoter sequence;
a nucleic acid sequence encoding a first protein of interest that is operably linked to the internal promoter; and
a poly A signal sequence,
wherein the nucleic acid construct further comprises at least one insertion element, and
wherein the expression construct insertion elements are compatible with the dock site insertion elements to facilitate insertion of the nucleic acid expression construct at the dock site.
133 . A method comprising:
providing a host cell of claim 1 ;
introducing into the host cell one or more nucleic acid expression constructs encoding a first protein of interest under conditions such that the nucleic acid expression constructs are inserted at the dock sites, wherein the nucleic acid expression construct further comprises at least the following elements in operable association in 5′ to 3′ order:
a selectable marker sequence;
an internal promoter sequence;
a nucleic acid sequence encoding the first protein of interest that is operably linked to the internal promoter; and
a poly A signal sequence,
wherein the nucleic acid construct further comprises at least one insertion element, and
wherein the expression construct insertion elements are compatible with the dock site insertion elements to facilitate insertion of the nucleic acid expression construct at the dock site.
134 - 184 . (canceled)