Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells

Edwards, Wesleigh F.; Young, Douglas D.; Deiters, Alexander

doi:10.1021/cb900041s

Cited by 86 publications

(86 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Laser excitation using photoconvertible fluorescent proteins, like Kikume-GR (Kulesa et al ., 2008; Nowotschin and Hadjantonakis, 2009), allows groups of cells to be tracked following exposure to discrete wavelengths, but the cytoplasmic fluorescence is rapidly diluted in rapidly dividing progenitors populations like NC derivatives. Photoactivatible forms of Cre that could indelibly mark cells subsequent to excitation (Edwards et al ., 2009) would greatly facilitate lineage tracing in the ENS and other NC derivatives where cells are closely juxtaposed. In the meantime, straightforward use of bright stable fluorescent alleles, like the Sox10 -H2BVenus we describe, offer access to initial processes of lineage segregation in the ENS and other NC derivatives.…”

Section: Discussionmentioning

confidence: 99%

Isolation and live imaging of enteric progenitors based on Sox10‐Histone2BVenus transgene expression

Corpening¹,

Deal²,

Cantrell³

et al. 2011

Genesis

View full text Add to dashboard Cite

To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type specific expression that reflects endogenous Sox10 patterns. Individual cells exhibiting nuclear–localized fluorescence of the H2BVenus reporter are readily visualized in both fixed and living tissue and are amenable to isolation by fluorescence activated cell sorting (FACS). FACS-isolated H2BVenus+ enteric NC-derived progenitors (ENPs) exhibit multi-potency, readily form neurospheres, self-renew in vitro and express a variety of stem cell genes. Dynamic live imaging as H2BVenus+ ENPs migrate down the fetal gut reveals cell fragmentation suggesting that apoptosis occurs at a low frequency during normal development of the ENS. Confocal imaging both during population of the fetal intestine and in post-natal gut muscle strips revealed differential expression between individual cells consistent with down-regulation of the transgene as progression towards non-glial fates occurs. The expression of the Sox10-H2BVenus transgene in multiple regions of the peripheral nervous system will facilitate future studies of NC lineage segregation as this tool is expressed in early NC progenitors and maintained in enteric glia.

show abstract

Section: Discussionmentioning

confidence: 99%

Isolation and live imaging of enteric progenitors based on Sox10‐Histone2BVenus transgene expression

Corpening¹,

Deal²,

Cantrell³

et al. 2011

Genesis

View full text Add to dashboard Cite

show abstract

“…Deiters and coworkers have answered this question with the development of a light-activated version of Cre (125). Using unnatural amino acid incorporation methodologies in E. coli, Tyr324 was replaced by o-nitrobenzyl tyrosine (ONBY), effectively blocking the active site nucleophile and rendering Cre inactive.…”

Section: Cre Transductionmentioning

confidence: 99%

Cre Recombinase

Duyne

2015

Microbiol Spectr

View full text Add to dashboard Cite

The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

show abstract

“…[4][5][6][7][8][9][10] For the generation of caged proteins in live cells, the genetic code can be expanded allowing for the site-specific incorporation of caged amino acids into proteins. [11][12][13][14][15][16][17][18][19][20] The caged amino acids are inserted in response to an amber stop codon, TAG, by suppressor tRNAs that have been misacylated by engineered tRNA synthetases. [21][22][23][24][25][26][27][28] Advantages of this methodology over other approaches are that the location of the unnatural amino acid is determined through genetic mutagenesis and the light-activated proteins are produced in live cells eliminating the requirement for transfection or injection.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

et al. 2013

Self Cite

View full text Add to dashboard Cite

Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the sitespecific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

show abstract

Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells

Cited by 86 publications

References 36 publications

Isolation and live imaging of enteric progenitors based on Sox10‐Histone2BVenus transgene expression

Isolation and live imaging of enteric progenitors based on Sox10‐Histone2BVenus transgene expression

Cre Recombinase

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

Contact Info

Product

Resources

About