TYROSINE PROTOTROPHY
Provided herein is a tyrosine selection marker system, and uses thereof. In some embodiments, nucleic acid constructs, vectors, host cells and related compositions and methods for generating and selecting tyrosine prototroph cells are provided.
1 . A recombinant nucleic acid construct comprising a nucleotide sequence of interest, and one or both of: i) a phenylalanine hydroxylase (PAH) gene, and ii) a pterin-4-alpha-carbinolamine dehydratase (PCBD1) gene.
2 . The recombinant nucleic acid construct of claim 1 , wherein the nucleic acid construct further comprises a recombination target sequence.
3 . The recombinant nucleic acid construct of claim 2 , wherein the recombination target sequence is a FLP Recognition Target (“FRT”), lox, or Bxb1 sequence.
4 . The recombinant nucleic acid construct of claim 1 , wherein the nucleotide sequence of interest encodes a polypeptide of interest, encodes an RNA molecule of interest, or contains a restriction enzyme site.
5 . The recombinant nucleic acid construct of claim 1 , wherein the nucleotide sequence of interest is a first nucleotide sequence of interest, and wherein the recombinant nucleic acid construct further comprises a second nucleotide sequence of interest.
6 . (canceled)
7 . (canceled)
8 . The recombinant nucleic acid construct of claim 5 , wherein the first nucleotide sequence of interest encodes a first polypeptide comprising an antibody variable light (VL) region and wherein the second nucleotide sequence of interest encodes a second polypeptide comprising an antibody variable heavy (VH) region.
9 . A vector comprising the recombinant nucleic acid construct of claim 1 .
10 . (canceled)
11 . (canceled)
12 . A host cell comprising the recombinant nucleic acid construct of claim 1 .
13 . A host cell comprising the recombinant nucleic acid construct of claim 1 , wherein the recombinant nucleic acid construct is stably integrated into a chromosome of the host cell.
14 . The host cell of claim 12 , wherein the host cell is a mammalian cell.
15 . The host cell of claim 14 , wherein the mammalian cell is a mouse cell, a human cell, or a Chinese hamster ovary (CHO) host cell.
16 . Use of a host cell of claim 12 for production of a polypeptide or RNA molecule encoded by the nucleotide sequence of interest.
17 . A recombinant polypeptide produced by the host cell of claim 12 .
18 . A composition comprising a host cell claim 12 , and a cell culture medium.
19 . A composition comprising a host cell, a recombinant nucleic acid construct of claim 1 , and a cell culture medium.
20 . (canceled)
21 . The composition of claim 18 , wherein the cell culture medium is tyrosine-deficient.
22 . The composition of claim 21 , wherein the cell culture medium comprises less than 1 mM tyrosine.
23 . (canceled)
24 . A method of producing a host cell comprising an exogenous nucleotide sequence of interest, the method comprising:
a) introducing into a host cell an exogenous nucleic acid construct comprising the nucleotide sequence of interest, wherein the exogenous nucleic acid construct further comprises: i) a PAH gene, and ii) a PCBD1 gene;
b) culturing the host cell comprising the exogenous nucleic acid construct in a tyrosine-deficient medium, wherein the host cell comprising the exogenous nucleic acid construct proliferates more rapidly in the tyrosine-deficient medium than a corresponding otherwise identical host cell that lacks the exogenous nucleic acid construct.
25 . The method of claim 24 , wherein the exogenous nucleic acid construct is stably integrated into a chromosome of the host cell.
26 . (canceled)
27 . A method of producing a host cell comprising a first exogenous nucleotide sequence of interest and a second exogenous nucleotide sequence of interest, the method comprising:
a) introducing into a host cell I) a first exogenous nucleic acid construct comprising i) the first exogenous nucleotide sequence of interest and ii) a PAH gene and II) a second exogenous nucleic acid construct comprising i) the second exogenous nucleotide sequence of interest and ii) a PCBD1 gene; and
b) culturing the host cell comprising the first exogenous nucleic acid construct and the second exogenous nucleic acid construct in a tyrosine-deficient medium, wherein the host cell comprising the first exogenous nucleic acid construct and the second exogenous nucleic acid construct proliferates more rapidly in the tyrosine-deficient medium than a corresponding otherwise identical host cell that lacks the first exogenous nucleic acid construct and second exogenous nucleic acid construct.
28 . The method of claim 27 , wherein the first exogenous nucleic acid construct and the second exogenous nucleic acid construct are both stably integrated into a first chromosome of the host cell, or wherein the first exogenous nucleic acid construct is stably integrated into a first chromosome of the host cell and the second exogenous nucleic acid construct is stably integrated into a second chromosome of the host cell.
29 . (canceled)
30 . (canceled)
31 . (canceled)
32 . The recombinant nucleic construct claim 1 , wherein at least one of:
i) the PAH gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or a sequence with at least 80% homology thereof;
ii) the PAH gene comprises a DNA sequence shown in SEQ ID NO: 2, or a sequence with at least 80% homology thereof;
iii) the PCBD1 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, or a sequence with at least 80% homology thereof; or
iv) the PCBD1 gene comprises a DNA sequence shown in SEQ ID NO: 4, or a sequence with at least 80% homology thereof.