Generation of a non-leaky heat shock-inducible Cre line for conditional Cre/lox strategies in zebrafish.
Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of the mammalian genome. Recently, we showed that Cre is also highly efficient in zebrafish and temporal control of recombination can be achieved by using the ligand-inducible CreER(T2). Previous attempts have been made to control recombination by using the temperature inducible hsp70l promoter to conditionally drive the expression of Cre or EGFP-Cre, respectively. However, in this study we demonstrate that the hsp70l promoter possesses a basal leakiness resulting in Cre-mediated recombination even at permissive temperatures. In order to prevent non-conditional recombination, we combined the hsp70l promoter with a mCherry-tagged ligand-inducible CreER(T2). At permissive temperatures and in the absence of the ligand tamoxifen (TAM), no non-conditional recombination is observed indicating tight regulation of CreER(T2). Instead, comprehensive site-specific recombination is mediated following heat induction and administration of TAM.
Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology, Dresden, Germany.
Stefan
S
Hans
Dorian
D
Freudenreich
Michaela
M
Geffarth
Jan
J
Kaslin
Anja
A
Machate
Michael
M
Brand
D000818:Animals; D030801:Animals, Genetically Modified; D004625:Embryo, Nonmammalian; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006358:Hot Temperature; D019426:Integrases / Q000235:genetics*; D008297:Male; D008954:Models, Biological; D011401:Promoter Regions, Genetic / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology*; D019076:Transgenes / Q000235:genetics / Q000502:physiology; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D018840:HSP70 Heat-Shock Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases