Homologous recombination in multipotent adult progenitor cells
View Patent ↗The invention relates to methods of altering gene expression by homologous recombination in a multipotent adult progenitor cell (MAPC). In particular, methods of producing a recombinant MAPC, of correcting a genetic defect in a mammal, of providing a functional and/or therapeutic protein to a mammal, and of transforming a MAPC are provided. MAPCs containing an erogenous DNA as well as recombinant MAPCs and their differentiated progeny are also provided.
1. A method of altering gene expression in a cell, the method comprising:
a) introducing, by nucleoporation, a polynucleotide into isolated expanded human non-embryonic, non-germ cells, with a transfection efficiency up to 14% and without toxicity to the cells, the cells having undergone at least 10-40 cell doublings in culture, wherein the cells are multipotent adult progenitor cells characterized in that they are non-embryonic, non-germ cells that express oct4, are not transformed, and have a normal karyotype, the polynucleotide comprising a sequence of interest; and
b) culturing the nucleoporated cells produced in step a) under conditions sufficient to express the sequence of interest, such that the resulting cells have altered gene expression.
2. The method of claim 1 wherein the polynucleotide also comprises a sequence homologous to a genomic DNA sequence in the cells and the polynucleotide is homologously recombined in the cells.
3. The method of claim 1 or 2 wherein the non-embryonic, non-germ cells further express one or more of telomerase, rex-1, rox-1, or sox-2.
4. The method of claim 1 or 2 wherein the non-embryonic, non-germ cells can differentiate into at least one cell type of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages.
5. The method of claim 4 wherein the non-embryonic, non-germ cells further express one or more of telomerase, rex-1, rox-1, or sox-2.
6. The method of claim 4 wherein the non-embryonic, non-germ cells can differentiate into at least one cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages.
7. The method of claim 6 wherein the non-embryonic, non-germ cells further express one or more of telomerase, rex-1, rox-1, or sox-2.
8. The method of claim 1 or 2 , further comprising combining the non-embryonic, non-germ cells produced in step b) with a pharmaceutically-acceptable carrier.
9. The method of claim 1 or 2 , wherein the non-embryonic, non-germ cells are cultured at a density of about 500 cells/cm 2 -1500 cells/cm 2 .
10. The method of claim 9 , wherein the non-embryonic, non-germ cells are cultured at a density of about 500 cells/cm 2 .
11. The method of claim 1 , wherein the non-embryonic, non-germ cells are isolated from bone marrow.