Establishment of Nile Tilapia Primary Cell Culture Methods and In Vitro Cell Knockdown Techniques

Abstract

As an important aquaculture species and research model, Nile tilapia (Oreochromis niloticus) has not yet been systematically studied for the isolation, culture, and in vitro gene manipulation techniques of primary cells from various tissues. This study aimed to explore methods for isolating primary cells from various tissues, as well as developing in vitro gene manipulation techniques in Nile tilapia. Four different Nile tilapia tissues were enzymatically digested and separated using trypsin or collagenase. Collagenase (0.1%) was used for the digestion of the gonads, liver, and heart, while trypsin (0.25%) showed better adhesion efficiency for spleen tissue. Moreover, we assessed EGFP fluorescence intensity and cell survival rates following transfection with empty siRNA (siRNA-NC), lentivirus (LV-NC), and six adeno-associated virus (AAV-NC) serotypes (AAV2-NC, AAV5-NC, AAV6-NC, AAV8-NC, AAV9-NC, AAV-DJ-NC) in gonadal cells. The results demonstrated that cells transfected with siRNA-NC and LV-NC showed the highest levels of green fluorescent protein expression and survival rates in primary gonadal cells, compared to AAC-NC. Subsequently, we knocked down the Kdm6bb gene in Nile tilapia primary gonadal cells by transfecting them with LV-Kdm6bb and siRNA-Kdm6bb. qPCR and immunofluorescence analyses demonstrated a significant reduction in Kdm6bb mRNA levels following transfection with siRNA-Kdm6bb compared to siRNA-NC, and with LV-Kdm6bb compared to LV-NC. This study offers valuable tools for the validation of primary cell isolation and in vitro molecular regulatory mechanisms and functions in Nile tilapia.