Human gdnf knock-in at the bovine β-casein locus by CRISPR/Cas9-mediated homologous recombination.

Parkinson's disease (PD) is a common and debilitating neurodegenerative disorder that stems from the loss of dopaminergic neurons and is associated with progressive motor dysfunction. Glial cellderived neurotrophic factor (GDNF) is very promising for the treatment of PD and other neuropathic diseases. In this study we applied the CRISPR/Cas9 technique to develop a gene-targeting knock-in system for the expression of the human gdnf gene at the bovine β-casein gene locus. A CRISPR/Cas9 expression plasmid and a pP40-GN vector were constructed. Bovine fetal fibroblasts were cultured and collected using the tissue explant method. Then the pP40-GN and CRISPR/Cas9 vectors were electrotransfected into bovine fetal fibroblasts. Resistant clones were selected using G418, while target clones were identified via PCR analyses and PCR product sequencing. Bovine fetal fibroblasts were successfully isolated and cultured using the ear tissue blocking method. After co-transfecting the bovine fetal fibroblasts with the pP40-GN target vector and the CRISPR/Cas9 expression vector in 7 days of selection with G418 a total of 12 healthy and well-separated cell clones were obtained, of which 5 were involved in gene targeting events. This study lays a foundation for the production of human GDNF protein using a gene-targeted bovine mammary gland bioreactor and provides a new strategy for the targeted therapy of PD. [ABSTRACT FROM AUTHOR]