Limits...
Split-Cre complementation restores combination activity on transgene excision in hair roots of transgenic tobacco.

Wen M, Gao Y, Wang L, Ran L, Li J, Luo K - PLoS ONE (2014)

Bottom Line: In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro 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.In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes.Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing, China; Chongqing Key Laboratory of Transgenic Plant and Safety Control, Southwest University, Chongqing, China.

ABSTRACT
The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. 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 in vitro 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.

Show MeSH
The in vivo recombination of split-Cre protein and the deletions determined by GUS activity.A: Digram of plant expression vectors. pCambia refer to vector of pCambia1305.1. B: GUS staining of transgenic hair roots for each transformant. “n” represents nuclear localization signal. The following are all the same.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4201524&req=5

pone-0110290-g002: The in vivo recombination of split-Cre protein and the deletions determined by GUS activity.A: Digram of plant expression vectors. pCambia refer to vector of pCambia1305.1. B: GUS staining of transgenic hair roots for each transformant. “n” represents nuclear localization signal. The following are all the same.

Mentions: We constructed a series of plant expression vectors for split-Cre complementation system (Fig. 2A). The plant expression vector pCAMBIA1305.1 [38], in which the E. coli gusA gene has been replaced by GUSPlus, served as an empty control. These recombinant plasmids carrying the split-Cre and full-length Cre genes were generated based on the pCAMBIA1305.1 vector. The gene cassette ploxP containing nos terminator sequences flanked by two 34-bp loxP sites in direct orientation, was used as a negative control. pCre and pnCre, containing full-length Cre and NLS-fused Cre driven by the CaMV 35S promoter served as positive controls. The schematic diagrams of all plant binary vectors were showed in Fig. 2A. The gene cassettes pNCre and pnNCre contained NCre and NLS-fused NCre, whereas pCCre and pnCCre contained CCre and NLS-fused CCre, respectively. The gene cassette pCCre-nNCre carried CCre and NLS-fused NCre. In the gene cassette pnCCre-nNCre, a NLS was fused into N terminus of the CCre and NCre genes, respectively.


Split-Cre complementation restores combination activity on transgene excision in hair roots of transgenic tobacco.

Wen M, Gao Y, Wang L, Ran L, Li J, Luo K - PLoS ONE (2014)

The in vivo recombination of split-Cre protein and the deletions determined by GUS activity.A: Digram of plant expression vectors. pCambia refer to vector of pCambia1305.1. B: GUS staining of transgenic hair roots for each transformant. “n” represents nuclear localization signal. The following are all the same.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4201524&req=5

pone-0110290-g002: The in vivo recombination of split-Cre protein and the deletions determined by GUS activity.A: Digram of plant expression vectors. pCambia refer to vector of pCambia1305.1. B: GUS staining of transgenic hair roots for each transformant. “n” represents nuclear localization signal. The following are all the same.
Mentions: We constructed a series of plant expression vectors for split-Cre complementation system (Fig. 2A). The plant expression vector pCAMBIA1305.1 [38], in which the E. coli gusA gene has been replaced by GUSPlus, served as an empty control. These recombinant plasmids carrying the split-Cre and full-length Cre genes were generated based on the pCAMBIA1305.1 vector. The gene cassette ploxP containing nos terminator sequences flanked by two 34-bp loxP sites in direct orientation, was used as a negative control. pCre and pnCre, containing full-length Cre and NLS-fused Cre driven by the CaMV 35S promoter served as positive controls. The schematic diagrams of all plant binary vectors were showed in Fig. 2A. The gene cassettes pNCre and pnNCre contained NCre and NLS-fused NCre, whereas pCCre and pnCCre contained CCre and NLS-fused CCre, respectively. The gene cassette pCCre-nNCre carried CCre and NLS-fused NCre. In the gene cassette pnCCre-nNCre, a NLS was fused into N terminus of the CCre and NCre genes, respectively.

Bottom Line: In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro 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.In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes.Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing, China; Chongqing Key Laboratory of Transgenic Plant and Safety Control, Southwest University, Chongqing, China.

ABSTRACT
The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. 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 in vitro 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.

Show MeSH