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. 2024 Oct 11;33(24):2094–2110. doi: 10.1093/hmg/ddae144

Figure 3.

Figure 3

CRE-lox-mediated reconstitution of large genes delivered by tripartite AAV vectors: Proof-of-concept. (A) Schematic representation of large gene reconstitution by CRE-lox mediated recombination of three AAV vector genomes. A gene-of-interest is split into three fragments (CDS1, 2, and 3) and delivered to target cells via three separate AAV vectors. For a proof-of-concept demonstration, IFT140-N (5′ 1923 bp of IFT140), BBS1, and LZTFL1 coding sequences were used as CDS1, 2, and 3, respectively. HA tag sequences were added to the 5′ ends of IFT140-N and BBS1. CRE recombinase was delivered via a separate AAV vector. SD: Splice donor site, SA: Splice acceptor site, 15: loxJT15, 17: loxJTZ17, and pA: polyA signal. (B) Production of IFT140N + BBS1 + LZTFL1 fusion proteins (red arrowheads) from a tripartite AAV vector set. AAV vectors depicted in panel (A) were transduced to 293T cells, and the expression of IFT140N + BBS1 + LZTFL1 fusion proteins was examined by SDS-PAGE and immunoblotting using HA and LZTFL1 antibodies. Numbers on the right mark the locations of protein standards. A separate AAV vector, AAV-EF1α-CRE, was co-transduced to express CRE (lanes 1–7). Lane 1: CDS1 (IFT140-N) only, lane 2: CDS2 (BBS1) only, lane 3: CDS3 (LZTFL1) only, lane 4: CDS1 + CDS2, lane 5: CDS1 + CDS3, lane 6: CDS2 + CDS3, lane 7: CDS1 + CDS2 + CDS3, lane 8: CDS1 + CDS2 + CDS3 (without CRE). Endogenous LZTFL1 (blue arrowheads) was used as a loading control.