Enhanced gene expression
The present disclosure is directed to a novel, unexpected approach of expressing exogenous gene(s) at increased levels by predictably, optionally irreversibly, incorporating the gene(s) into a region of increased gene expression (RIDGE) on a chromosome of a host cell. This approach is accomplished by identification of RIDGE(s) and further by integration of integrase-specific sites (e.g., attP or attB) in the presence of or mediated by integrase. The approach renders a high level of gene expression; predictability of the location of the exogenous genes, elimination of genetic instability or unwanted phenotype; and reduction of time and cost in optimizing protein production in host cells, which will be every useful in the production of therapeutic, prophylactic, and diagnostic proteins.
1. An isolated host cell comprising
an exogenous gene flanked by a first integrase-resulting site and a second integrase-resulting site; and
a fraction of green fluorescent protein (GFP) gene sequence,
wherein the exogenous gene and the fraction of GFP gene sequence are located at a region of increased gene expression (RIDGE) on a chromosome of the cell, and wherein the fraction of GFP gene sequence does not express GFP.
2. The isolated host cell of claim 1 , wherein the first integrase-resulting site is attL.
3. The isolated host cell of claim 1 , wherein the second integrase-resulting site is attR.
4. An isolated host cell comprising
a first exogenous gene flanked by a set of first integrase-resulting sites;
a second exogenous gene flanked by a set of second integrase-resulting sites; and
a fraction of GFP gene sequence,
wherein each of the first exogenous gene, the second exogenous gene and the fraction of GFP gene sequence is located at a RIDGE in the host cell, and wherein the fraction of GFP gene sequence does not express GFP.
5. The isolated host cell of claim 4 , wherein the set of the first integrase-resulting sites is the same or not the same as the set of the second integrase-resulting sites.
6. The isolated host cell of claim 4 , wherein the first and second exogenous genes are located at a single RIDGE.
7. The isolated host cell of claim 4 , wherein the first exogenous gene is located at a first RIDGE and the second exogenous gene is located at a second RIDGE.
8. The isolated host cell of claim 7 , wherein the first and second RIDGEs are in a single chromosome.
9. The isolated host cell of claim 7 , wherein the first RIDGE is in a first chromosome and the second RIDGE is in a second chromosome.