Generation of transgenic marmosets expressing genetically encoded calcium indicators

Abstract

Chronic monitoring of neuronal activity in the living brain with optical imaging techniques became feasible owing to the continued development of genetically encoded calcium indicators (GECIs). Here we report for the first time the successful generation of transgenic marmosets (Callithrix jacchus), an important nonhuman primate model in neurophysiological research, which were engineered to express the green fluorescent protein (GFP)-based family of GECIs, GCaMP, under control of either the CMV or the hSyn promoter. High titer lentiviral vectors were produced, and injected into embryos collected from donor females. The infected embryos were then transferred to recipient females. Eight transgenic animals were born and shown to have stable and functional GCaMP expression in several different tissues. Germline transmission of the transgene was confirmed in embryos generated from two of the founder transgenic marmosets that reached sexual maturity. These embryos were implanted into six recipient females, three of which became pregnant and are in advanced stages of gestation. We believe these transgenic marmosets will be invaluable non-human primate models in neuroscience, allowing chronic in vivo monitoring of neural activity with functional confocal and multi-photon optical microscopy imaging of intracellular calcium dynamics.

Generation of genetically engineered non‐human primate models of brain function and neurological disorders

“…Much of the initial work to adopt and adapt genetic and gene-editing tools to NHP was done in macaques (Chan, 2013; Chan et al, 2001). However, marmosets offer several advantages as an ideal NHP species for the development of genetically engineered lines (Izpisua Belmonte et al, 2015; Okano & Kishi, 2018; Okano, Miyawaki, & Kasai, 2015), in particular, an intergeneration time and establishment of transgenic lines two-three times faster than in macaques (Park et al, 2016; Sasaki et al, 2009). This is as important a factor as the continued development of efficient genetic tools adapted to NHP.…”

“…In this article, we first review the present state of genetic engineering techniques as applied to NHP, from the early efforts to induce exogeneous gene expression in macaques (Chan, Chong, Martinovich, Simerly, & Schatten, 2001; Chan et al, 2000; Niu et al, 2015; Niu et al, 2010; Yang et al, 2008), to the latest results in producing germline transmission of different transgenes (Liu et al, 2016; Park et al, 2016; Putkhao et al, 2013; Sasaki et al, 2009) and the establishment of knockout lines of specific genes (Y. Chen et al, 2017; Y.…”

“…Liu et al, 2014; Niu et al, 2014; Sato et al, 2016; Wan et al, 2015; Zuo et al, 2017). We then report our own efforts to generate transgenic marmosets expressing genetically-encoded calcium indicators (Park et al, 2016), and talk about our preliminary efforts to generate a marmoset model of CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). We conclude with some future perspectives on the further development of genetic tools for generation of genetically engineered NHP.…”

“…After the initial technical breakthrough in making GFP-expressing transgenic marmosets (Sasaki et al, 2009), we reported marmosets expressing functional reporter genes (Park et al, 2016), while another group reported a marmoset model of polyglutamine (polyQ) disease (Tomioka, Ishibashi, et al, 2017; Tomioka, Nogami, et al, 2017). The transgenic marmosets expressing functional reporter genes were generated by our own efforts and the summary of our work and ongoing progress will be discussed later in this review.…”

“…Recently, we generated transgenic marmosets expressing GCaMP under the regulation of either ubiquitous (CMV) or neuron-specific (hSyn1) promoters by lentivirus based transgenesis (Park et al, 2016). The GCaMP protein is one of the GFP-based genetically encoded calcium indicators, which works as a visible marker of cellular calcium dynamics, allowing monitoring of neuronal activity in vitro and in vivo (T.…”