Cloning of the zebrafish dβh promoter (5.2 kb) and creation of dβh:EGFP and dβh:MYCN lines have been previously described (13 (link)). Similarly, mCherry and human c-MYC cDNAs were PCR cloned into the pENTR223 vector, and dβh:mCherry and dβh:c-MYC constructs were individually assembled by Multisite Gateway cloning (Invitrogen). Using a co-injection strategy with I-SceI meganuclease, one-cell embryos were injected with DNA constructs and grown to adulthood. Primary injectants were screened for germline transmission, and stable lines were generated by outcrossing to the wild-type AB strain.
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Generating Transgenic Zebrafish Lines
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Corresponding Organization : Massachusetts Institute of Technology
Other organizations : Mayo Clinic
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Variable analysis
independent variables
- Cloning of the zebrafish dβh promoter (5.2 kb)
- Creation of dβh:EGFP and dβh:MYCN lines
- PCR cloning of mCherry and human c-MYC cDNAs into the pENTR223 vector
- Assembly of dβh:mCherry and dβh:c-MYC constructs by Multisite Gateway cloning
- Co-injection strategy with I-SceI meganuclease
- Injection of DNA constructs into one-cell embryos
- Germline transmission of primary injectants
- Generation of stable lines by outcrossing to the wild-type AB strain
- Wild-type AB strain
- Negative control: Not specified
- Positive control: Not specified
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