Protein production and protein delivery

A method of making a homologously recombinant cell in vitro wherein the expression of a targeted gene not normally expressed in the cell is altered, comprising the steps of:

• (a) transfecting the cell in vitro with a DNA construct, the DNA construct comprising:
• (i) a targeting sequence;
• (ii) a regulatory sequence;
• (iii) an exon;
• and
• (iv) an unpaired splice donor site, thereby producing a transfected cell;
• and
• (b) maintaining the transfected cell in vitro under conditions appropriate for homologous recombination.

A method of altering the expression of a targeted gene in a cell in vitro wherein the gene is not normally expressed in the cell, comprising the steps of:

• (a) transfecting the cell in vitro with a DNA construct, the DNA construct comprising:
• (i) a targeting sequence;
• (ii) a regulatory sequence;
• (iii) an exon;
• and
• (iv) an unpaired splice donor site, thereby producing a transfected cell;and
• (b) maintaining the transfected cell in vitro under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell;
• and
• (c) maintaining the homologously recombinant cell in vitro under conditions appropriate for expression of the gene.

A method of making a protein by altering the expression of a targeted gene in a cell in vitro wherein the gene is not normally expressed in the cell, comprising the steps of:

• a) transfecting the cell in vitro with a DNA construct, the DNA construct comprising:
• (i) a targeting sequence;
• (ii) a regulatory sequence;
• (iii) an exon;
• and
• (iv) an unpaired splice donor site, thereby producing a transfected cell;
• (b) maintaining the transfected cell in vitro under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell;
• and
• c) maintaining the homologously recombinant cell in vitro under conditions appropriate for expression of the gene thereby making the protein.