A reliable and flexible gene manipulation strategy in posthatch zebra finch brain

Abstract

Songbird models meaningfully contribute to many fields including learned vocal communication, the neurobiology of social interactions, brain development, and ecology. The value of investigating gene-brain-behavior relationships in songbirds is therefore high. Viral infections typically used in other lab animals to deliver gene editing constructs have been less effective in songbirds, likely due to immune system properties. We therefore leveraged the in vivo electroporation strategy used in utero in rodents and in ovo in poultry, and apply it to posthatch zebra finch songbird chicks. We present a series of experiments with a combination of promoters, fluorescent protein genes, and piggyBac transposase vectors to demonstrate that this can be a reliable, efficient, and flexible strategy for genome manipulation. We discuss options for gene delivery experiments to test circuit and behavioral hypotheses using a variety of manipulations, including gene overexpression, CRISPR/Cas9 gene editing, inducible technologies, optogenetic or DREADD cellular control, and cell type-specific expression.

Development of an Acute Method to Deliver Transgenes Into the Brains of Adult Xenopus laevis

“…Electroporation has been successfully used to deliver exogenous genes into the brains of a variety of species in vivo ( Wang and Mei, 2013 ; Ahmadiantehrani and London, 2017 ), including X. laevis tadpoles ( Haas et al, 2001 , 2002 ). Recently, a method has been described to introduce exogenous genes into a restricted region of the adult brains of zebrafish, greatly enhancing the utility of the technique ( Zou et al, 2014 ).…”

Gene manipulation to test links between genome, brain and behavior in developing songbirds: a test case

“…In vivo electroporation side-steps issues of viral infection and is highly effective for transgene delivery in zebra finches ( Fig. 3; Ahmadiantehrani and London, 2017b). Electroporation takes advantage of the fact that DNA is negatively charged and that electrical current disrupts cell membranes so that plasmids can enter cells.…”

“…Because songbirds are altricial, the procedure can be performed on post-hatching chicks, which show >95% survival, and of these >95% show transgene expression ( Fig. 3; Ahmadiantehrani and London, 2017b). Addition of a transposase such as piggyBac effectively integrates cargo into the genome, and brain cells then express the transgene through to at least P50, permitting genetic manipulation in the context of questions of maturation and tutor song memorization (Ahmadiantehrani and London, 2017b).…”

“…3; Ahmadiantehrani and London, 2017b). Addition of a transposase such as piggyBac effectively integrates cargo into the genome, and brain cells then express the transgene through to at least P50, permitting genetic manipulation in the context of questions of maturation and tutor song memorization (Ahmadiantehrani and London, 2017b). Of note, piggyBac transposase-integrated transgenes can also be excised from the genome (Woodard and Wilson, 2015).…”

AAV1 is the optimal viral vector for optogenetic experiments in pigeons (Columba livia)

“…Viral vector efficiency can vary considerably between brain areas and species highlighting the need for viral transfection studies in various model organisms 34 – 39 . Especially in birds, viral transfection has proven to be difficult 40 , possibly due to properties of the immune system 41 , 42 . Although optogenetics has been already used in some areas of the zebra finch song system 7 , 43 , 44 , the efficiency of different viral constructs was not compared within these and other brain areas such as the visual system.…”