Characterization of the Properties of Seven Promoters in the Motor Cortex of Rats and Monkeys After Lentiviral Vector-Mediated Gene Transfer

Abstract

Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken β-actin [CAG], human elongation factor-1α [EF-1α], and Rous sarcoma virus [RSV]) and two cell type-specific promoters (rat synapsin I and mouse α-calcium/calmodulin-dependent protein kinase II [CaMKIIα]) in rat and monkey motor cortices in vivo were characterized. Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of the various promoters were prepared and injected into rat and monkey motor cortices. Immunohistochemical analysis revealed that all of the VSV-G-pseudotyped lentiviral vectors had strong endogenous neuronal tropisms in rat and monkey brains. Among the seven promoters, the CMV promoter showed modest expression in glial cells (9.4%) of the rat brain, whereas the five ubiquitous promoters (MSCV, CMV, CAG, EF-1α, and RSV) showed expression in glial cells (7.0-14.7%) in the monkey brain. Cell type-specific synapsin I and CaMKIIα promoters showed excitatory neuron-specific expression in the monkey brain (synapsin I, 99.7%; CaMKIIα, 100.0%), but their specificities for excitatory neurons were significantly lower in the rat brain (synapsin I, 94.6%; CaMKIIα, 93.7%). These findings could be useful in basic and clinical neuroscience research for the design of vectors that efficiently deliver and express transgenes into rat and monkey brains.

Avian sarcoma leukosis virus receptor-envelope system for simultaneous dissection of multiple neural circuits in mammalian brain

“…Lentiviral vectors were produced by cotransfection of HEK293T cells with four plasmids (Table S3) and titrated as described (25,26). In in vitro experiments, HEK293T cells were serially infected by the FuGB2 vector and the Env vector (3 d after FuGB2 vector infection).…”

“…In in vivo experiments, ten-week-old rats were serially injected with the FuGB2 vector (somatosensory cortex) and the Env vectors (thalamus, 3 wk after FuGB2 vector injections). Injected rats were then perfused with 4% (wt/vol) paraformaldehyde and their brains were processed for histological analyses as described (26). All data are presented as mean ± SE (SEM).…”

“…In the present study, this vector was used to express ASLV receptors in both in vitro and in vivo experiments. The cytomegalovirus (CMV) promoter was selected because it drives a high level of protein expression in the mammalian brain (26) and has been used previously for TVA expression (8,21,27). We tested all possible receptor-envelope combinations by serially infecting receptor-expressing and Env-pseudotyped vectors into HEK 293T cells.…”

Viral Vectors for Delivering Gene Material into Cells and Their Application in Neurobiology (Review)

“…In laboratory practice, lentiviral vectors with synapsin promoter contributing to the expression of the given gene in any type of neuronal cells are used [114][115][116][117]. Lentiviral vectors are also used with the promoter containing calcium/calmodulin-dependent protein kinase II alpha with the expression in the exciting glutamatergic neurons [118,119].…”

Gene Therapy for the Nervous System: Challenges and New Strategies

“…The majority of these studies have used strong ubiquitous promoters to provide a broad overview of transgene expression in different tissues and cell types throughout the body after intravascular infusions, or CNS cell types after intracranial delivery. The CAG promoter (also referred to as CBA and CB) is the promoter most commonly used in these types of studies as it is demonstrably a strong promoter that mediates long-term gene expression in CNS and other tissues [103]. This hybrid promoter cassette was designed originally with the cytomegalovirus immediate early enhancer fused to the chicken β-actin promoter that includes 300 bp of the upstream region and a portion of intron 1 because it carries a strong enhancer conserved across species [104].…”