Targeted integration into stem cells
Disclosed herein are methods and compositions for targeted integration of sequences of interest such as lineage-specific or cell fate reporter constructs or protein encoding sequences into stem cells.
1. An isolated pluripotent or multipotent stem cell comprising a transgene, wherein the transgene is integrated into an endogenous PITX3 or Factor IX gene of the stem cell using a pair of zinc finger nucleases that bind to and cleave the endogenous PITX3 or Factor IX gene, wherein the pair of zinc finger nucleases bind to paired target sites of SEQ ID NO:47 and SEQ ID NO:48 of the PITX3 gene, or paired target sites of SEQ ID NO:49 and SEQ ID NO:50 of the PITX3 gene, or to paired target sites of SEQ ID NO:51 and SEQ ID NO:56 of the Factor IX gene, and further wherein the pair of zinc finger nucleases cleaves the PITX3 or Factor IX gene within or between the paired target sites such that the transgene is integrated into the PITX3 or Factor IX gene.
2. The isolated stem cell of claim 1 , wherein the stem cell is selected from the group consisting of a hematopoietic stem cell, a mesenchymal stem cell, an embryonic stem cell, a neuronal stem cell, a muscle stem cell, a liver stem cell, a skin stem cell, and an induced pluripotent stem cell.
3. The isolated stem cell of claim 1 , wherein the stem cell is a mammalian stem cell.
4. The isolated stem cell of claim 3 , wherein the stem cell is a human induced pluripotent stem cell (hiPSC).
5. The isolated stem cell of claim 1 , wherein the transgene is flanked by recombinase sites.
6. The isolated stem cell of claim 1 , further comprising a sequence encoding a reporter construct.
7. A stem cell according to claim 1 , wherein the isolated transgene comprises a sequence encoding a lineage-specific or cell fate marker as shown in Table 1.