title: Intravital fluorescence imaging of small interfering RNA-mediated gene repression in a dual reporter melanoma xenograft model.
abstract: Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control. Intratumoral injection of a siRNA specific to the incorporated siRNA target site, complexed with a cationic lipid that has been optimized for in vivo delivery, resulted in 65%±11% knockdown of tdTFP relative to EGFP quantified by in vivo imaging and 68%±10% by reverse transcription-quantitative polymerase chain reaction. No effect was observed with nonspecific control siRNA treatment. This model provides a platform on which siRNA delivery technologies can be screened and optimized in vivo.
authors.affiliation: TransDerm Inc., Santa Cruz, California, USA.
authors.forename: Robyn P
authors.initials: RP
authors.lastname: Hickerson
authors.forename: Emilio
authors.initials: E
authors.lastname: Gonzalez-Gonzalez
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Vlassov
authors.forename: Mu
authors.initials: M
authors.lastname: Li
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Lara
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Contag
authors.forename: Roger L
authors.initials: RL
authors.lastname: Kaspar
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D009368:Neoplasm Transplantation; D061848:Optical Imaging; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology*
chemical_list: D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases