Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
Disclosed herein are methods and compositions for inactivating a FUT8 gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.
1. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:
F1: QSSDLSR (SEQ ID NO: 9);
F2: TSGNLTR (SEQ ID NO: 10);
F3: RSDDLSK (SEQ ID NO: 11); and
F4: DRSALAR (SEQ ID NO: 12).
2. An isolated polynucleotide encoding the zinc finger DNA-binding domain of claim 1 .
3. A fusion protein comprising a zinc finger DNA-binding domain according to claim 1 and at least one cleavage domain or at least one cleavage half-domain.
4. The fusion protein of claim 3 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
5. The fusion protein of claim 4 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.
6. A polynucleotide encoding the fusion protein according to claim 3 .
7. An isolated cell comprising the fusion protein according to claim 3 .
8. The cell of claim 7 , wherein Fut8 is partially inactivated.
9. The cell of claim 7 , wherein Fut8 is fully inactivated.
10. The cell of claim 7 , wherein one or more additional genes are partially or fully inactivated.
11. The cell of claim 10 , wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.
12. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises five zinc finger recognition regions ordered F1 to F5 from N-terminus to C-terminus, and wherein F1, F2, F3, F4, and F5 comprise the following amino acid sequences:
FI: RSDNLRE (SEQ ID NO: 26);
F2: NNTQLIE (SEQ ID NO: 27);
F3: TSSILSR (SEQ ID NO: 28);
F4 RSDNLSA (SEQ ID NO: 29); and
F5: RKDTRIT (SEQ ID NO: 30).
13. An isolated polynucleotide encoding the zinc finger DNA-binding domain of claim 12 .
14. A fusion protein comprising a zinc finger DNA-binding domain according to claim 12 and at least one cleavage domain or at least one cleavage half-domain.
15. The fusion protein of claim 14 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
16. The fusion protein of claim 14 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.
17. A polynucleotide encoding the fusion protein according to claim 14 .
18. An isolated cell comprising the protein according to claim 14 .
19. The cell of claim 18 , wherein Fut8 is partially inactivated.
20. The cell of claim 18 , wherein Fut8 is fully inactivated.
21. The cell of claim 18 , wherein one or more additional genes are partially or fully inactivated.
22. The cell of claim 21 , wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.