Efficient method for production of compound transgenic animals

A method of producing a compound chimeric mouse, comprising:

• (a) obtaining mouse embryonic stem (ES) cells whose genomes' comprise a construct comprising a nucleic acid sequence encoding a marker gene and a gene of interest each of which is operatively linked to a promoter;
• (b) selecting said ES cells of (a) that functionally express said marker gene;
• (c) cloning said ES cells of (b) to obtain individual ES cell clones;
• (d) analyzing the site of integration of each of said individual ES cell clones of (c);
• (e) selecting at least two of said individual ES cell clones of (d) with different sites of integration;
• (f) mixing said individual ES cell clones selected in (e) to form a mixed pool of ES cell clones with different integration sites;
• (g) co-culturing said mixed pool of ES cell clones with a mouse morula having a zona pellucida removed such that compound chimeric mouse embryos are formed;
• (h) transferring said compound chimeric mouse embryos to a pseudopregnant female;
• and
• (i) obtaining a compound chimeric mouse comprising cells that express said gene of interest to detectable levels.