A new protocol for functional analysis of adipogenesis using reverse transfection technology and time-lapse video microscopy.
Since the worldwide increase in obesity represents a growing challenge for healthcare systems, research focusing on fat cell metabolism has become a focal point of interest. Here, we describe a small interfering RNA (siRNA)-technology-based screening method to study fat cell differentiation in human primary preadipocytes that could be further developed towards an automated middle-throughput screening procedure. First, we established optimal conditions for the reverse transfection of human primary preadipocytes demonstrating that an efficient reverse transfection of preadipocytes is technically feasible. Aligning the processes of reverse transfection and fat cell differentiation utilizing peroxisome proliferator-activated receptor gamma (PPAR gamma)-siRNA, we showed that preadipocyte differentiation was suppressed by knock-down of PPAR gamma, the key regulator of fat cell differentiation. The use of fluorescently labelled fatty acids in combination with fluorescence time-lapse microscopy over a longer period of time enabled us to quantify the PPAR gamma phenotype. Additionally, our data demonstrate that reverse transfection of human cultured preadipocytes with TIP60 (HIV-1 Tat-interacting protein 60)-siRNA lead to a TIP60 knock-down and subsequently inhibits fat cell differentiation, suggesting a role of this protein in human adipogenesis. In conclusion, we established a protocol that allows for an efficient functional and time-dependent analysis by quantitative time-lapse microscopy to identify novel adipogenesis-associated genes.
Grönniger
Elke
E
Research and Development, Research Special Skincare, Beiersdorf AG, Unnastrasse 48, 20245 Hamburg, Germany.
Wessel
Sonja
S
Kühn
Sonja Christin
SC
Söhle
Jörn
J
Wenck
Horst
H
Stäb
Franz
F
Winnefeld
Marc
M
D017667:Adipocytes / Q000166:cytology / Q000502:physiology*; D050156:Adipogenesis / Q000502:physiology*; D005260:Female; D051548:Histone Acetyltransferases / Q000235:genetics; D006801:Humans; D000074443:Lysine Acetyltransferase 5; D018715:Microscopy, Video / Q000379:methods*; D047495:PPAR gamma / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D047495:PPAR gamma; D034741:RNA, Small Interfering; D051548:Histone Acetyltransferases; C098489:KAT5 protein, human; D000074443:Lysine Acetyltransferase 5