Journal: bioRxiv

Article Title: Neuronal cell line expressing full-length mutant huntingtin exhibits alterations in proteolysis

doi: 10.64898/2026.01.15.699723

Figure Lengend Snippet: Application of the Sleeping Beauty system for the transposition of normal/mutant huntingtin genes into the genome of Neuro-2a cells allows to create HD transgenic cellular model and determine the levels of HttQ15/Q138 genes expression in obtained cell lines. a) The validation of transgenes expression by quantitative PCR (qPCR). SB::Q15/Q138 – pSB tet -Neo vector containing HttQ15 or HttQ138 gene, SB::(-) – pSB tet -Neo vector without huntingtin gene insertion, -SB – Neuro-2a cells without transfection, RT – reverse transcription; b) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression; c) Western blotting of poly/monoclonal transgenic Neuro-2a cells lysates without/with doxycycline induction of HttQ15 or HttQ138 expression. Data for control Neuro-2a lines (SB::(-), with transfection of «empty» vector, and -SB, without transfection, are represented also. Top panel – lysates of four different monoclonal transgenic Neuro-2a lines with HttQ15 expression, middle panel – same as top, but for lysates of HttQ138 monoclonal lines, bottom panel – lysates of polyclonal transgenic Neuro-2a lines; d) The transgene (HttQ15/HttQ138) copy number in the genomes of monoclonal (Q15m/Q138m) and polyclonal (Q15p/Q138p) Neuro-2a lines. The GAPDH-normalized Htt gene copy number in the SB::(-) control line was referred to 1. e) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression, (f) Western blotting of lysates without/with doxycycline induction of HttQ15 or HttQ138 expression after 3 years of storage, cultivation and passaging. Total protein content in each sample was controlled by β-actin immunoreactivity. Data in Tables b) and f) and Fig. d) are represented as mean ± S.D. (n = 3).

Article Snippet: After pSB tet -Neo PCR linearization (using Q5® High-Fidelity DNA Polymerase, New England Biolabs, USA) with introduction of MluI and NotI restriction sites (forward primer with NotI site: 5′-AATA GCGGCC GCGCTTCCATCGATAGACATGATAAGATAC-3′, reverse primer with MluI site: 5′-TATT ACGCGT CAGAGGCCTTTCGAGGGTAGG-3′; restriction sites are underlined) as well as restriction of the pCI-HttQ15 and pCI-HttQ138 plasmids at MluI and NotI sites, the resulted fragments were ligated (insertion:vector = 1:3 in molar ratio) and transformation of competent E. coli XL1-Blue cells with the obtained ligase mixture was carried out.

Techniques: Mutagenesis, Transgenic Assay, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Reverse Transcription, Western Blot, Control, Passaging