Stem cells modified to facilitate threonine catabolism
View Patent ↗A mammalian cell comprising a recombinant, functional L-threonine 3-dehydrogenase (EC 1.1.1.103; TDH) gene, methods of making, and methods of use.
1. A mammalian stem cell not in a human and comprising a recombinant, functional L-threonine 3-dehydrogenase (EC 1.1.1.103; TDH) gene, wherein:
the TDH gene is a transgene;
the TDH gene comprises an endogenous-to-the-cell TDH allele operably-linked to a heterologous-to-the-cell transactivator, wherein expression of the gene is activated by an exogenous-to-the-cell activator;
the TDH gene is heterologous to the cell;
the TDH gene is human, wherein human gene mutations at the AG to GG splice acceptor mutations in exons 4 and 6, as well as the non-sense mutation within exon 6 wherein arginine codon 214 is replaced by a translational stop codon, are corrected to impart L-threonine 3-dehydrogenase catalytic activity to the L-threonine 3-dehydrogenase encoded by the TDH gene; or
the TDH gene is over-expressed, as compared with a corresponding wild-type TDH gene, and comprises a coding or transcription regulatory sequence heterologous to the cell.
2. A mammalian stem cell not in a human and comprising a recombinant, functional L-threonine 3-dehydrogenase (EC 1.1.1.103; TDH) gene, wherein the TDH gene is a transgene.
3. A mammalian stem cell not in a human and comprising a recombinant, functional L-threonine 3-dehydrogenase (EC 1.1.1.103; TDH) gene, wherein the TDH gene comprises an endogenous-to-the-cell TDH allele operably-linked to a heterologous-to-the-cell transactivator, wherein expression of the gene is activated by an exogenous-to-the-cell activator.
4. The mammalian stem cell of claim 1 , wherein the TDH gene is inactivatable by a Cre-lox system, wherein the gene is flanked by loxP sites, and the cell further comprises a gene encoding Cre recombinase.
5. The mammalian stem cell of claim 2 , wherein the TDH transgene is a mouse TDH transgene.
6. A mammalian stem cell not in a human and comprising a recombinant, functional L-threonine 3-dehydrogenase (EC 1.1.1.103; TDH) gene, wherein the TDH gene is human, wherein human gene mutations at the AG to GG splice acceptor mutations in exons 4 and 6, as well as the non-sense mutation within exon 6 wherein arginine codon 214 is replaced by a translational stop codon, are corrected to impart L-threonine 3-dehydrogenase catalytic activity to the L-threonine 3-dehydrogenase encoded by the TDH gene.
7. A mammalian stem cell not in a human and comprising a recombinant, functional L-threonine 3-dehydrogenase (EC 1.1.1.103; TDH) gene, wherein the TDH gene is over-expressed, as compared with a corresponding wild-type TDH gene, and comprises a coding or transcription regulatory sequence heterologous to the cell.
8. The mammalian stem cell of claim 1 , wherein the cell is a primary explant cell.
9. The mammalian stem cell of claim 1 , wherein the mammalian stem cell is an embryonic stem cell.
10. A cell culture comprising the mammalian stem cell of claim 1 .
11. The mammalian stem cell of claim 1 , wherein the TDH gene is a mouse TDH gene and the cell is human and ex vivo.
12. The mammalian stem cell of claim 2 , wherein the mammalian stem cell is an embryonic stem cell.
13. The mammalian stem cell of claim 3 , wherein the mammalian stem cell is an embryonic stem cell.
14. The mammalian stem cell of claim 5 , wherein the mammalian stem cell is an embryonic stem cell.
15. The mammalian stem cell of claim 6 , wherein the mammalian stem cell is an embryonic stem cell.
16. The mammalian stem cell of claim 7 , wherein the mammalian stem cell is an embryonic stem cell.