RESUMO
We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit. For complete details on the use and execution of this protocol, please refer to Matsushita et al.1.
Assuntos
Roedores , Animais , Ratos , TransgenesRESUMO
The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.
Assuntos
Vetores Genéticos , Recombinação Genética , Transgenes , Vetores Genéticos/genética , GenomaRESUMO
BACKGROUND: Pseudotyping of a lentiviral vector with fusion glycoproteins composed of rabies virus glycoprotein (RVG) and vesicular stomatitis virus glycoprotein (VSVG) segments achieves high gene transfer efficiency through retrograde transport in the nervous system. In our previous study, we determined the junction of RVG/VSVG segments of glycoproteins that enhances the transduction efficiency of the neuron-specific retrograde gene transfer (NeuRet) vector (termed fusion glycoprotein type E or FuG-E). NEW METHOD: We aimed to optimize the amino acid residue at position 440 in the membrane-proximal region of FuG-E to improve the efficiency of retrograde gene transfer in the brain. RESULTS: We constructed variants of FuG-E with 18 kinds of single amino acid substitutions at residue 440 to generate lentiviral vectors pseudotyped with these variants, and tested in vivo gene transfer of the vectors in the mouse brain. The FuG-E (P440E) variant, in which proline was substituted by glutamate at residue 440 in FuG-E, showed the greatest retrograde gene transfer efficiency in the brain, bearing the property of the NeuRet vector. The FuG-E (P440E) pseudotype also displayed efficient retrograde gene transfer in the common marmoset brain. COMPARISON WITH EXISTING METHODS: The NeuRet vector with the FuG-E (P440E) variant demonstrated higher retrograde gene transfer efficiency into different neural pathways compared with the parental FuG-E vector. CONCLUSIONS: The FuG-E (P440E) pseudotype provides a powerful tool to investigate neural circuit mechanisms underlying various brain functions and for gene therapy trials of neurological and neurodegenerative diseases.