Flp-mediated recombination
Compositions and methods useful for homologous recombination and stable integration of a polynucleotide into a host cell are provided. The disclosed compositions and methods provide a rapid and efficient method for stably integrating an exogenous polynucleotide into a host cell and selecting such transformed cells.
1 . A recombination cassette comprising
a promoter/enhancer region;
a polynucleotide of interest;
a polyA signal domain; an FRT recombination domain; and
a dhfr polynucleotide,
wherein the promoter/enhancer region, the polynucleotide of interest and the polyA signal domain are operably linked.
2 . The recombination cassette of claim 1 , wherein the promoter/enhancer region comprises a human CMV immediate early 1 (hCMV IE1).
3 . The recombination cassette of claim 2 , wherein the hCMV IE 1 promoter/enhancer region comprises a sequence as set forth from about x 1 to about x 2 of SEQ ID NO:1 or 2, wherein x 1 is a nucleotide from position 1 to position 70 and x 2 is a nucleotide from position 770 to position 780.
4 . The recombination cassette of claim 1 , farther comprising a variable length intervening sequence (VLIVS) comprising a splice donor site and a splice acceptor site.
5 . The recombination cassette of claim 4 , wherein the VLIVS comprises an intron A of a hCMV IE1 gene.
6 . The recombination cassette of claim 4 , wherein the VLIVS comprises an intron A of a hCMV IE1 gene that has a deletion between the splice donor site and splice acceptor site of the intron A.
7 . The recombination cassette of claim 6 , wherein the VLIVS comprises a sequence from about x 3 to about x 4 of SEQ ID NO:1, wherein x 3 is a nucleotide from 770-780 and x 4 is a nucleotide from 1300-1310 of SEQ ID NO:1; or from about x 5 to about x 6 of SEQ ID NO:2, wherein x 5 is a nucleotide from 770-780 and x 6 is a nucleotide from 1300-1310 of SEQ ID NO:2.
8 . The recombination cassette of claim 1 , wherein the polynucleotide of interest encodes a therapeutic agent.
9 . The recombination cassette of claim 1 , wherein the polyA signal domain comprises at least 100 contiguous nucleotides of SEQ ID NO:3.
10 . The recombination cassette of claim 9 , wherein the polyA signal domain comprises SEQ ID NO:3.
11 . A recombination vector comprising a recombination cassette of claim 1 .
12 . The recombination vector of claim 11 , further comprising
a second promoter/enhancer region;
a second polynucleotide of interest; and
a second polyA signal domain,
wherein the second promoter/enhancer region, the second polynucleotide of interest, and the second polyA signal are operably linked.
13 . The recombination vector of claim 12 , further comprising an intervening domain between the second promoter/enhancer region and the second polynucleotide of interest.
14 . A host cell comprising a recombination vector of claim 11 .
15 . The host cell of claim 14 , wherein the host cell is adapted for growth in suspension.
16 . The host cell of claim 14 , wherein the host cell is adapted for growth in serum-free medium.
17 . The host cell of claim 15 , wherein the host cell is adapted for growth in serum-free medium.
18 . A host cell comprising a recombination cassette of claim 1 .
19 . The host cell of claim 18 , wherein the host cell is adapted for growth in suspension.
20 . The host cell of claim 18 , wherein the host cell is adapted for growth in serum-free medium.
21 . The host cell of claim 19 , wherein the host cell is adapted for growth in serum-free medium.
22 . A recombination system comprising:
a recombination cassette of claim 1; and
a host cell comprising an FRT site.
23 . The recombination system of claim 22 , wherein the host cell is a CHO cell.
24 . The recombination system of claim 23 , wherein the CHO cell is a CHO-DG44 cell.
25 . The recombination system of claim 22 , wherein the host cell is adapted for growth in suspension.
26 . The recombination system of claim 22 , wherein the host cell is adapted for growth in serum-free medium.
27 . The recombination system of claim 22 , wherein the host cell is derived from a CHO-DG44 cell.
28 . The recombination system of claim 22 , wherein the host cell is dhfr − .
29 . A kit comprising a vector of claim 10 and a host cell comprising an FRT site.
30 . The kit of claim 29 , wherein the host cell is a dhfr CHO host cell, the genome of which comprises an FRT site.