Fig 1: DdCBE-mediated KO of rat mtProteins in C6 cells.(A) Strategy of mtDNA-encoded protein depletion by DdCBE-introduced stop codon. The W codon TGA was changed to TAA stop codon (W stop). The Q codon CAA was changed to TAA stop codon (Q stop). (B) Location of introduced stop codon at mtProteins. (C to L) The identification of DdCBE-mediated mtProtein depletion in ND2-KO (C), ND3-KO (D), ND4-KO (E), ND5-KO (F), ND6-KO (G), CYTB-KO (H), COX1-KO (I), COX2-KO (J), COX3-KO (K), and ATP6-KO (L) C6 cells by deep sequencing (left), Western blot (middle), and Seahorse assay (right). For deep sequencing, n = 3 technical replicates. For Seahorse assay, n = 5 technical replicates for Ctrl and n = 6 technical replicates for each KO cell line. Data were presented as means ± SD.
Fig 2: A cKO rat resource for mtProteins.(A to D) Genotyping of LSL-DdCBE-Nd2 KI (A), LSL-DdCBE-Nd5 KI (B), LSL-DdCBE-Cytb KI (C), and LSL-DdCBE-Cox3 KI (D) F0 rats by PCR with upstream primer pair (Up band), downstream primer pair (Down band), and internal primer pair (DddAtox band). (E and F) The recombination sites were confirmed by Sanger sequencing for upstream primer pair (E) and downstream primer pair (F) in LSL-DdCBE-Nd2 KI, LSL-DdCBE-Nd5 KI, LSL-DdCBE-Cytb KI, and LSL-DdCBE-Cox3 KI rats. (G) Six cKO rat strains covering four respiratory chain complexes were generated in this study.
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