Development of high-concentration lipoplexes for in vivo gene function studies in vertebrate embryos

Abstract

Here we report that highly concentrated cationic lipid/helper lipid‐nucleic acid complexes (lipoplexes) can facilitate reproducible delivery of a variety of oligonucleotides and plasmids to chicken embryos or to mouse embryonic mesenchyme. Specifically, liposomes composed of N,N‐dioleyl‐N,N‐dimethylammonium chloride (DODAC)/1,2 dioleoyl glycero‐3‐phosphorylethanolamine (DOPE) prepared at 18‐mM concentrations produced high levels of transfection of exogenous genes in vivo and in vitro. Furthermore, we report sufficient uptake of plasmids expressing interference RNA to decrease expression of both exogenous and endogenous genes. The simplicity of preparation, implementation, and relatively low toxicity of this transfection reagent make it an attractive alternative for developmental studies in post‐gastrulation vertebrate embryos. Developmental Dynamics 240:2108–2119, 2011. © 2011 Wiley‐Liss, Inc.

Avian Facial Morphogenesis Is Regulated by c-Jun N-terminal Kinase/Planar Cell Polarity (JNK/PCP) Wingless-related (WNT) Signaling

“…Fibroblasts expressing either RCAS::WNT11 (44) or RCAS::GFP (obtained from Stephen J. Gaunt) were injected into the presumptive maxillary region of stage 15-17 embryos (46). Embryos were reincubated for 48 h, processed either for whole mount in situ hybridization, immunofluorescence, BrdU, or TUNEL staining as described (11,34,47). For skeletal analysis, embryos were incubated until stage 38 and stained for cartilage and bone as described (48).…”

“…The shWNT11 plasmid was obtained from others (49), and the shGFP targeting construct was cloned in our laboratory following published sequence (34,50). Liposomes composed of 18 mM DODAC/DOPE were mixed with shRNA plasmids or empty pRFPRNAi vector to create lipoplexes.…”

“…The lipoplex mixture was injected into the maxillary prominence at stage 18, and embryos were grown for 24 -30 h until stage 23-24. Embryos were processed into wax for BrdU staining (34).…”

“…Mesenchymal cells were transfected with various DNA constructs using DODAC/DOPE liposomes (34). Super Topflash plasmid was used to measure ␤-catenin-mediated Wnt activity (54) or ATF2-luc to measure PCP/JNK activity (55).…”

“…This mouse model will no doubt be crossed into many mutant lines in the future. The chicken embryo face is accessible during stages of lip fusion and can be transfected with plasmids using liposomes (34). It is thus possible to combine local manipulations of signaling pathways with these transfection methods to study the effect on cell behavior.…”