Multifunctional Lentiviral Plasmid Construction

The pcDNA5 GASTM-3-RVG-10-Lamp2b-HA plasmid was obtained from Addgene (#71295) and the GASTM-RVG cDNA sequence was replaced by the glycosylation sequence GNSTM followed by BbsI restriction sites using PCR and subsequent NEBuilder HiFi DNA assembly (New England Biolabs) according to manufacturer’s instructions. Afterward, the open-reading frame containing an N-terminal signal peptide (SP) was cloned into the pJET1.2/blunt cloning vector (Thermo Scientific) by PCR and NEBuilder HiFi DNA assembly, after which specific targeting peptide (TP) tags flanked by BbsI restriction sites were incorporated into the BbsI cut by T4 DNA ligase (New England Biolabs) according to manufacturer’s instructions. Complete SP-GNSTM-3-TP-10-Lamp2b-HA cDNA sequences were amplified by PCR to contain BamHI and NotI overhangs and cloned into the pHAGE2-EF1alpha-IRES-PuroR-WPRE lentiviral vector (Wilson et al., 2008 (link); de Jong et al., 2020 (link)). TP tags included: TP-FLAG (DYKDDDDK), TP1 (RGD-4C; ACDCRGDCFCG), TP2 (CRPPR) and TP-9R (RRRRRRRRR). 3; 10: glycine-serine amino acid spacers. Oligo sequences are provided in Supplementary Table S1. Primer sequences are available upon request.

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Roefs M.T., Heusermann W., Brans M.A., Snijders Blok C., Lei Z., Vader P, & Sluijter J.P. (2022). Evaluation and manipulation of tissue and cellular distribution of cardiac progenitor cell-derived extracellular vesicles. Frontiers in Pharmacology, 13, 1052091.

Publication 2022

NEBuilder HiFi DNA assembly pJET1.2/blunt cloning vector T4 DNA ligase

Amino acid Cdna Glycine Glycosylation Ires Oligo Peptide Plasmid Primer Serine Signal peptide T4 dna ligase Vector

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Variable analysis

independent variables

Replacement of GASTM-RVG cDNA sequence with the glycosylation sequence GNSTM followed by BbsI restriction sites
Incorporation of specific targeting peptide (TP) tags flanked by BbsI restriction sites into the BbsI cut

dependent variables

Expression of SP-GNSTM-3-TP-10-Lamp2b-HA cDNA sequences in the pHAGE2-EF1alpha-IRES-PuroR-WPRE lentiviral vector

control variables

Maintenance of the open-reading frame containing an N-terminal signal peptide (SP)
Cloning of complete SP-GNSTM-3-TP-10-Lamp2b-HA cDNA sequences into the pHAGE2-EF1alpha-IRES-PuroR-WPRE lentiviral vector

controls

Positive control: None specified
Negative control: None specified

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