Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1) and electroporation: methods and optogenetic applications

Abstract

The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1) or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications.

The Effect of Millisecond Pulsed Electric Fields (msPEF) on Intracellular Drug Transport with Negatively Charged Large Nanocarriers Made of Solid Lipid Nanoparticles (SLN): In Vitro Study

“…The EP parameters (500 V/cm; 5 imp. each of 1.5 ms duration) for SLNs delivery were selected as optimal and non-toxic according to our MTT results and the available literature reports (Čemažar et al 2002 ; Yu et al 2011 ; Zou et al 2014 ; Huang et al 2014 ). Our results confirm the published data and indicate that no significant cytotoxic effect was observed in the case of both cell lines.…”

Cre-Controlled CRISPR (3C) Mutagenesis: Fast and Easy Conditional Gene Inactivation in Zebrafish

“…However, also exogenously delivered Cre activity is possible. Potential approaches might include in vivo electroporation of Cre expressing plasmids or Cre transgene delivery via herpes simplex type I viruses into different organs at various developmental and adult stages (Cerda et al, 2006;Zou et al, 2014). We used Cre mRNA injections at the 1-cell stage to elicit recombination at the earliest time point possible which resulted in pigmentation defects in both neural crest-derived melanocytes and anterior neural plate-derived RPE cells.…”

Minimally invasive brain injections for viral-mediated transgenesis: New tools for behavioral genetics in sticklebacks

“…Three promoters were piloted to drive gene expression based on work in zebrafish (Zou et al 2014) – a long-term promoter (hCMV, N = 43, Figure 2) resulting in fluorescent signal 2-5 weeks after injection, a short-term promoter (mCMV, N = 10) with expression between 4 and 7 days post-injection, and a retrograde promoter (hEF1a, N = 7) which did not result in a detectable fluorescent signal. Promoters were tested for their ability to drive a fluorescent protein (EGFP, EYFP, GCaMP6f, or mCherry).…”

“…To enable direct manipulation of these candidate genes and thereby examine how they contribute to behavior, we developed a neurosurgical method to deliver either pharmacological agents or transgenic elements directly into the stickleback brain. There is a dearth of information on surgical methodology in small (3-4 cm) fish, necessitating a refining of the anesthesia process (Neiffer & Stamper 2009; Sladky & Clarke 2016) and building a custom surgical rig (Zou et al 2014). To maximize animal welfare, we additionally needed to identify clear warning signs of failure to recuperate by establishing a normal recovery pattern in stickleback similar to the work in koi by Harms et al, (2005).…”