Methods and compositions for use of non-coding RNA in cell culturing and selection
Described herein are methods and compositions for producing a gene of interest (GOI) which, in certain embodiments, can reduce the metabolic burden on cells and reduce decoupling of GOI production from marker production, as compared to prior art methods. The methods relate to positive selection and negative selection approaches to establishing high GOI-producing cell lines, e.g., CHO lines. In certain embodiments, the methods comprise transfecting a cell with (a) an oligonucleotide comprising a GOI and a non-coding RNA, and (b) an oligonucleotide encoding a selection protein; wherein the non-coding RNA promotes or inhibits production of the selection protein. The cell producing the GOI can be identified and/or selected as a result of or by detecting the absence or the presence of the selection protein.
1. A method for identifying a cell producing a protein encoded by a gene of interest (GOI), the method comprising:
(1) transfecting the cell with
(a) a first oligonucleotide comprising the GOI and one or more non-coding RNA, and
(b) a second oligonucleotide encoding a selection protein, wherein the one or more non-coding RNA promotes or inhibits production of the selection protein, wherein the second oligonucleotide encoding a selection protein further comprises an sxRNA; (i) wherein the sxRNA is stabilized by the one or more non-coding RNA to promote translation of the selection protein or (ii) wherein the sxRNA comprises secondary structure that is disrupted by the one or more non-coding RNA to inhibit translation of the selection protein; and
(2) detecting the absence or the presence of the selection protein to identify the cell producing the protein encoded by the GOI.
2. The method of claim 1 , wherein the one or more non-coding RNA promotes cell survival and/or the amount of protein encoded by the GOI produced per cell.
3. The method of claim 1 , wherein the one or more non-coding RNA is ebv-miR-BART10-3p or mirR-7.
4. The method of claim 1 , comprising transfecting the cell with a oligonucleotide encoding a second selection protein, wherein the second selection protein is a lethal protein and wherein the one or more non-coding RNA inhibits production of the lethal protein.
5. The method of claim 1 , wherein the cell is a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) cell.
6. The method of claim 1 , wherein the selection protein is an antibiotic resistance protein, a fluorescent marker, or a cell surface marker, or a lethal protein.
7. The method of claim 1 , wherein the one or more non-coding RNA is encoded in an intron of the GOI.
8. The method of claim 1 , further comprising transfecting the cell with a second GOI and a second non-coding RNA, wherein the second GOI and the second non-coding RNA are located on the same oligonucleotide.
9. The method of claim 8 , wherein the second non-coding RNA is encoded in an intron of the second GOI.
10. The method of claim 8 , wherein the first and second non-coding RNAs are the same.
11. The method of claim 8 , wherein
(1) the first and second non-coding RNAs are different and wherein
(2) the oligonucleotide encoding the selection protein further encodes a second selection protein, or
wherein the method comprises transfecting the cell with a second oligonucleotide encoding a second selection protein, wherein
(2) the second non-coding RNA promotes or inhibits translation of the second selection protein.
12. A method for promoting survival of cells producing a protein encoded by a gene of interest (GOI), the method comprising:
transfecting cells with (a) an oligonucleotide comprising the GOI and one or more siRNA, and (b) an oligonucleotide encoding a protein lethal to the cells; wherein the one or more siRNA inhibits production of the protein lethal to the cells to promote survival of the cells producing the protein encoded by the GOI.
13. A method for identifying a cell producing a protein encoded by a gene of interest (GOI), the method comprising:
(1) transfecting one or more cells with
(a) a first oligonucleotide comprising a gene of interest (GOI) and one or more non-coding RNA, and
(b) a second oligonucleotide encoding a selection protein, wherein the one or more non-coding RNA is an miRNA or an siRNA that binds and inhibits translation of the second oligonucleotide encoding a selection protein; and
(2) identifying or selecting the cell producing the protein encoded by the GOI by detecting the absence of the selection protein.
14. The method of claim 13 , wherein the cell is a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) cell.
15. The method of claim 13 , wherein the selection protein is a fluorescent marker, a cell surface marker, or a lethal protein.
16. The method of claim 13 , wherein the one or more non-coding RNA is encoded in an intron of the GOI.
17. The method of claim 13 , wherein the selection protein is a lethal protein.
18. The method of claim 13 , further comprising: transfecting the cell with an oligonucleotide encoding a second selection protein, wherein the second selection protein is a lethal protein and wherein the one or more non-coding RNA inhibits expression of the lethal protein.
19. The method of claim 13 , further comprising: transfecting the cell with a second GOI and a second non-coding RNA, wherein the second GOI and the second non-coding RNA are located on the same oligonucleotide.