Development of human cells with RXFP1 knockdown using retroviral delivery of microRNA against human RXFP1

To study the specifi c actions of relaxin through RXFP1 in human cells, it would be advantageous to develop cell populations with permanent RXFP1 knockdown (KD). We have developed and assessed four microRNA against human RXFP1. One of the four designed microRNA displayed signifi cant RXFP1 KD as assessed by reduced relaxin binding when co-transfected with human RXFP1 into HEK-293T cells. The selected microRNA sequence was subsequently retrovirally delivered into the human dermal fi broblast cell line BJ3 which natively expresses RXFP1. The RXFP1 KD BJ3 cells displayed diminished RXFP1 mRNA expression and complete loss of ability of relaxin treatment to reduce collagen deposition after TGF-β1 stimulation. The retroviral expression of miRNA to successfully silence RXFP1 expression is an invaluable tool to investigate receptor specifi city, signalling and possible off -target eff ects of newly developed relaxin analogs