POLYNUCLEOTIDES WITH SELECTION MARKERS
A polynucleotide with a selection marker sequence under the control of a desirable HSVMin promoter leads to improved efficiency for biotechnology applications. The polynucleotide may, for example, be a transposon for use with Tc1/mariner systems. Through various embodiments of the present disclosure, one is able to efficiently and effectively introduce cargo regions from polynucleotides such as those carrying a glutamine synthetase gene into a host's DNA.
1 . A polynucleotide comprising:
a selection cassette, wherein the selection cassette comprises:
an HSVMin promoter sequence and a selection marker sequence, wherein the HSVMin promoter sequence is from 120 to 160 nucleotides long and comprises;
(a) SEQ ID NO: 14 or SEQ ID NO: 24,
(b) sequence complementary to SEQ ID NO: 14 or SEQ ID NO: 24,
(c) a sequence comprising at least 80% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 24 or
a sequence comprising at least 80% sequence identity to a sequence complementary to SEQ ID NO: 14 or SEQ ID NO: 24 over a span of at least 100 nucleotides.
2 . The polynucleotide of claim 1 , further comprising one or more expression cassettes.
3 . The polynucleotide of claim 2 , wherein the polynucleotide comprises a cargo region comprising the selection cassette and the one or more expression cassettes.
4 . The polynucleotide of claim 2 , wherein the one or more expression cassettes are oriented in a first direction and the selection cassette is oriented in a second direction, wherein the first direction and the second direction are opposite directions.
5 . The polynucleotide of claim 2 , wherein the one or more expression cassettes comprise a first expression cassette and a second expression cassette, wherein the first expression cassette and the second expression cassette are oriented in a first direction.
6 . (canceled)
7 . The polynucleotide of claim 5 , wherein the selection cassette is located between the first expression cassette and the second expression cassette.
8 . The polynucleotide of claim 1 , wherein the selection marker sequence is a glutamine synthetase sequence, and wherein the glutamine synthetase sequence codes for a glutamine synthetase protein.
9 . The polynucleotide of claim 5 , further comprising a third expression cassette.
10 . The polynucleotide of claim 3 , wherein the polynucleotide is a vector.
11 . The polynucleotide of claim 10 , wherein the vector is a transposon vector, a retroviral vector or a lentiviral vector.
12 .- 13 . (canceled)
14 . The polynucleotide of claim 10 , wherein the vector is a transposon vector and the cargo region is flanked by a sequence or a pair of sequences that are recognized by a Tc1/mariner transposase.
15 . The polynucleotide of claim 14 , wherein the Tc1/mariner transposase protein is a Sleeping Beauty transposase.
16 .- 19 . (canceled)
20 . The polynucleotide of claim 5 , wherein either:
(a) the first expression cassette comprises a nucleotide sequence that codes for an antibody heavy chain and the second expression cassette comprises a nucleotide sequence that codes for an antibody light chain; or
(b) the first expression cassette comprises a nucleotide sequence that codes for an antibody light chain and the second expression cassette comprises a nucleotide sequence that codes for an antibody heavy chain.
21 .- 28 . (canceled)
29 . The polynucleotide of claim 8 , wherein the glutamine synthetase sequence comprises or is complementary to SEQ ID NO: 26 and the HSVMin promoter sequence comprises or is complementary to SEQ ID NO: 14 or SEQ ID NO: 24.
30 .- 42 . (canceled)
43 . A genetic delivery system comprising the transposon vector of claim 11 and either a transposase protein, a DNA plasmid, or an mRNA, wherein the DNA plasmid or the mRNA encode the transposase protein.
44 . A method for integrating an exogenous nucleotide sequence into a nucleotide sequence in a host cell, said method comprising; introducing into the host cell the polynucleotide of claim 1 .
45 .- 52 . (canceled)
53 . A method for integrating an exogenous nucleotide sequence into a nucleotide sequence in a host cell, said method comprising; introducing into the host cell the polynucleotide of claim 1 , under conditions that allow for random integration.
54 . A cell comprising the polynucleotide of claim 1 .
55 .- 60 . (canceled)
61 . A method of generating a biologic material, a therapeutic protein or a non-protein therapeutic biologic, said method comprising culturing the cell of claim 54 .
62 .- 68 . (canceled)
69 . A kit for generating and modifying a protein, wherein the kit comprises the polynucleotide of claim 1 , wherein the polynucleotide is a first polynucleotide, and the kit further comprises a second polynucleotide,
wherein the first polynucleotide comprises at least one expression cassette that comprises a nucleotide sequence that codes for a polypeptide, and
wherein the second polynucleotide comprises a post-translational modification cassette, and the post-translational modification cassette codes for an amino acid sequence of a protein that is capable of a post-translational modification of the polypeptide coded by the first polynucleotide.
70 .- 85 . (canceled)