Morpholino-mediated gene knockdown in mammalian organ culture.
We examined the role of semaphorin3a in ureteric bud (UB) branching morphogenesis using mouse metanephric organ culture [Tufro et al. (Mech Dev 125:558-568, 2008)]. In vitro UB injection of Sema3a antisense morpholino resulted in increased branching morphogenesis. Cellular and tissue uptake of oligonucleotides was facilitated by a peptide-mediated method. Our findings were validated by in vitro translation and in Sema3a null mice. This chapter describes a method to perfuse the UB lumen with fluorescein-labeled oligonucleotides bound to a peptide carrier.
Department of Pediatrics and Nephrology, Yale University School of Medicine, New Haven, CT, USA. alda.tufro@yale.edu
Alda
A
Tufro
D000595:Amino Acid Sequence; D000818:Animals; D004337:Drug Carriers / Q000737:chemistry; D055785:Gene Knockdown Techniques / Q000379:methods*; D007668:Kidney / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D060172:Morpholinos / Q000008:administration & dosage* / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000235:genetics; D009924:Organ Culture Techniques / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D010477:Perfusion / Q000379:methods; D040121:Semaphorin-3A / Q000235:genetics*
D004337:Drug Carriers; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D010455:Peptides; D040121:Semaphorin-3A