Split-Cre Complementation Restores Combination Activity on Transgene Excision in Hair Roots of Transgenic Tobacco

<div><p>The Cre/loxP system is increasingly exploited for genetic manipulation of DNA <i>in vitro</i> and <i>in vivo</i>. It was previously reported that inactive ‘‘split-Cre’’ fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the <i>in vitro</i> recombination reaction in which only the N-terminal domain (NCre) of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre) or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS) was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.</p></div

DNA oligo sequences utilizes in this report.

<p>DNA oligo sequences utilizes in this report.</p

Digram of the split-Cre model and <i>in vitro</i> recombination of Split-Cre protein.

<p><b>A: Digram of the split-Cre model.</b> The intact Cre was designed to be split at the 60th amino acid residue. Two molecules of split-Cre were named NCre and CCre respectively. <b>B: Structure of the substrate catalyzed by purified protein. C: Recombination assay of Split-Cre protein </b><b><i>in vitro</i></b><b>.</b> M: DL5000 Marker; Plasmid: 2µl plasmid (90 ng/µl) of pLoxp-Ic-CCre629. The plasmid was respectively digested by <i>Hin</i>dIII and <i>Bam</i>HI (H+B), split protein NCre (NCre), split protein CCre (CCre), combination of split protein NCre and CCre (NCre + CCre), intact protein Cre (Cre) and MBP. Plasmid and MBP were used as negative control, H+B digestions were used as positive control. MBP tag was used to purify fusion proteins.</p

The <i>in vivo</i> recombination of split-Cre protein and the deletions determined by GUS activity.

<p><b>A: Digram of plant expression vectors.</b> pCambia refer to vector of pCambia1305.1. <b>B: GUS staining of transgenic hair roots for each transformant.</b> “n” represents nuclear localization signal. The following are all the same.</p

Analysis of CCre and NCre transcription in transgenic hairy roots. Semi-quantitative RT-PCR analysis of the transcription level of CCre or NCre in transformants carrying pnCCre or pnNCre (A) and pCCre-nNCre or pnCCre-nNCre (B).

<p>Tobacco 18S was used as an internal control. Total RNA was isolated from roots. Numbers represent the different lines of each recombinant.</p

The recombinant splite-Cre excises DNA fragment between two Loxp sties <i>in vivo</i>. Vallidation of the non-excision (A) and excision (B) of DNA fragment in hairy roots.

<p>The amplified fragments of non-excision was 862 bp and 664bp for pNCre, pnNCre and pCCre, pnCCre, respectively. The amplified fragments of post-excision was 369 bp, while the pre-excision was 862 bp and 664 bp for pCCre-nNCre, pnCCre-nNCre and pCre, pnCre, respectively. M: DL2000 Marker; P, pre-excision signal; E, post-excision signal. pCambia and ploxP were used as control. <b>C: Schematic illustration of deletion in pCCre-nNCre and the sequencing result after deletion of DNA fragment.</b></p

Additional file 1 of A novel peptide PDHK1-241aa encoded by circPDHK1 promotes ccRCC progression via interacting with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway

Additional file 1: Table S1. Association between clinicopathological characteristic and circPDHK1 expression in ccRCC patients in validation phase. Table S2. Clinicopathological features of 4 patients with ccRCC in circRNA microarray. Table S3. Information of shRNAs and siRNAs. Table S4. Information of RNA probes. Table S5. Sequences of primers. Table S6. The differentially expressed circRNAs in ccRCC tissues compared to matched noncancerous tissues with WGCNA analysis. Table S7. Candidate differentially-expressed circRNAs. Table S8. Candidate binding proteins detected by Flag-IP and LC-MS. Table S9. Association between clinicopathological characteristic and HIF-2A expression in ccRCC patients in validation phase