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. Author manuscript; available in PMC: 2016 Feb 10.
Published in final edited form as: Integr Biol (Camb). 2015 Feb 10;7(2):237–249. doi: 10.1039/c4ib00221k

Figure 3. Correcting Aberrant Splicing of the Human CD22 Gene Using Splicesome-mediated RNA Trans-splicing Technology.

Figure 3

[A-C] RT-PCR analyses of ALL-1 and RAJI cells transfected with CD22 RTM vs. empty plasmid (EPL). Total RNA for the PCR was extracted 2d or 4 d after transfection with the CD22 RTM or EPL. [A] depicts an agarose gel that shows the RT-PCR amplification of a 415-bp RTM-specific RNA-segment amplified by RT-PCR using a CD22 exon 11 primer as forward primer (EX11-F) and the RTM IRES primer RTM-4494-5263 as a reverse primer (IRES-R), thereby confirming successful transfection of ALL-1 and RAJI cells with the CD22 RTM. [B] Successful completion of selective CD22 RTM-induced trans-splicing in ALL-1 cells was confirmed by specifically amplifying a 763-bp trans-spliced mRNA segment that includes segments of both ALL1-derived exon 9 and RTM-derived sequences via RT-PCR with an exon 9 forward primer (EX9-F) and a reverse primer in the IRES sequence of the RTM (IRES-R), which is just downstream of exon 14. hCD22E9-Fwd 5′-CAACCCTGACCTGTGAGAGC-3′/IRES-R 5′-AAGCGGCTTCGGCCAGTAAC-3′. Depicted is an agarose gel showing the RT-PCR products. [C] shows an agarose gel of the RT-PCR products obtained with the P10 primer set (EX4-F/EX4-R) that was used as a positive control of RNA integrity to amplify a 213-bp region (c.433–c.645) within Exon 4 of the CD22 cDNA. [D] Western blot analyses of ALL-1 and RAJI cells transfected with CD22 RTM vs. empty plasmid (EPL). [D1] and [D2] depict the anti-CD22 Western blots of proteins extracted from ALL-1 and RAJI cells 4d after transfection with CD22-RTM or EPL. In A-D, untransfected ALL-1 and RAJI cells also served as controls. CD22ΔE12 depletion causes cell death via apoptosis. Therefore, accurate RT-PCR and Western blot assays are not feasible beyond 4 d post transfection with the CD22-RTM.