A Preliminary Study Of The Transfection Of STOP Lentiviral Vector Into Cultured Cortical Neurons Of BTBR Mouse Model Of Autism

Objective:The aim of this study is to construct over-expressing lentiviral vector of stable tubule only polypeptide(STOP)and lentiviral vector without exogenous gene by multisite Gateway technology and to establish a simple method to culture high density cortical neurons of BTBR mouse.The results will give clue to explore the possible mechanism of STOP improving autism-like behavior of BTBR mouse,and to provide theoretical and experimental basis for clinical application in autism.Methods:1.The expression vector of pLV[Exp]-mCherry:T2A:Puro-EF1A>STOP was constructed by multisite Gateway clone technique.The lentiviral particles were prepared by transient co-transfection of the expression vector and auxiliary plasmids into 293T cells.Then the virus titer were measured,and the recombination were verified by observing fluorescence expression under fluorescence microscope after transfecting293T cells.The lentivirus vector without exogenous gene was constructed by the same method.2.The bilateral cerebral cortex of embryonic BTBR mice was used for culture in this study.Meninges was stripped,the cerebral cortex was ful y chopped up and digested by pancreatin.Then the cels were incubated into 6-well plates coated with poly-D-lysincoated.The morphological characteristics of neuronal at different stages were observed.Results:1.The STOP lentiviral vector of pLV[Exp]-mCherry:T2A:Puro-EF1A>STOP was successfully constructed,confirmed using DNA sequencing.The packed lentivirus vector had high efficiency transfection and the virus titer is approximately 5×10~7TU/mL.The mCherry could be observed after transfecting 293T cells.The lentivirus vector without exogenous gene of pLV[Exp]-CMV>mCherry(ns):T2A:Puro was successfully constructed by the same method.2.The cortical neurons of BTBR mouse were growing wel,which had morphological characteristics at different stages.After 1 day,the neurons adhered to the wall and small protrusions appeared.After 3 days,the neurons continued to extend protrusions and sparse neural networks appeared.After 7 days,deep and wide neural networks formed.Conclusion:1.The over-expressing lentiviral vector of stable tubule only polypeptide(STOP)and lentiviral vector without exogenous gene were successfully constructed.2.The cortical neurons of BTBR mouse model of autism were successfully cultured.