title: Construction of bicistronic lentiviral vectors for tracking the expression of CDNF in transduced cells.
abstract: CDNF is a recently described evolutionary conserved neurotrophic factor reported to be of relevance for the treatment of Parkinson's disease. Treatment with recombinant CDNF showed neurorestorative and neuroprotective effects on dopaminergic neurons in Parkinsonian animal models. Similar results are obtained using adeno-associated viral (AAV) vectors for CDNF expression in these animal models; however, the extent of the transduced brain tissue is difficult to assess due to the lack of reporter genes in the vectors used. Here, we describe two bicistronic lentiviral plasmids based on the Δ1D/2A and IRES elements for the expression of EGFP and rat CDNF, in order to track the transduced cells expressing CDNF with EGFP fluorescence. Transfected heterologous cells or transduced neurons with these vectors are easily identified by EGFP fluorescence and CDNF expression results in its recruitment to the endoplasmic reticulum (ER) by both bicistronic vectors. CDNF immunostaining is also observed in the Golgi apparatus when expressed in heterologous cells or hippocampal neuronal cultures; however, colocalization with a dense core secretory vesicle marker was scarce. Additionally, we showed that the expression of CDNF inhibited dendrite formation in hypothalamic neurons, suggesting that CDNF expressed by these bicistronic lentiviral vectors is functional and could have a role in neuronal morphology. The bicistronic lentiviral plasmids developed here could be of use to study the effect of rat CDNF at the cellular level or to better delineate the perikarya of neurons transduced with lentiviral vectors in animal models of Parkinson's disease.
authors.affiliation: Laboratory of Neurobiology, Department of Biomedicine, Faculty of Allied Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.
authors.forename: Anllely
authors.initials: A
authors.lastname: Fernández
authors.affiliation: Laboratory of Neurobiology, Department of Biomedicine, Faculty of Allied Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.
authors.forename: Sheyla
authors.initials: S
authors.lastname: Guzmán
authors.affiliation: Laboratory of Neurobiology, Department of Biomedicine, Faculty of Allied Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile.
authors.forename: Yocelin
authors.initials: Y
authors.lastname: Cruz
authors.affiliation: Laboratory of Neurobiology, Department of Biomedicine, Faculty of Allied Health Sciences, Universidad de Antofagasta, Antofagasta 1270300, Chile. Electronic address: zamorano@gmail.com.
authors.forename: Pedro
authors.initials: P
authors.lastname: Zamorano
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016086:Lentivirus / Q000235:genetics*; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000502:physiology; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C577430:CDNF protein, rat; D009414:Nerve Growth Factors; D011994:Recombinant Proteins
keywords: Bicistronic; CDNF; EGFP; Lentivirus; Neurons; Parkinson's disease