Development of a Hair Follicle Reconstitution Model utilizing Cashmere Goat Hair Follicle Stem Cells

Cashmere is a rare animal-derived fiber with substantial economic value. Here, we established a hair follicle reconstitution model using secondary hair follicle stem cells from Cashmere goats (gSHFSCs). This model enables direct assessment of reconstructed fiber readouts-including length, diameter, and yield-thereby offering a tractable framework for functional interrogation of candidate genes implicated in cashmere-associated traits. To enhance assay performance and reproducibility, we systematically optimized key parameters, including the input cell number and the dermal cell-to-gSHFSC ratio. In parallel, we defined electroporation conditions that support efficient delivery of either plasmid DNA or Cas9 ribonucleoprotein (RNP) complexes into gSHFSCs. We implemented two gene-disruption strategies that achieved knockout efficiencies exceeding 90%. Finally, we incorporated gSHFSC knockout lines targeting selected genes into the reconstitution model, which elicited distinct phenotypic shifts in reconstructed fiber characteristics (P < 0.05). Collectively, these results indicate that our hair follicle reconstitution model constitutes a robust approach for validating candidate genes relevant to economically important cashmere traits.