D. Genetically Modified Organisms

Glofish^® are the colorful green, red, orange,blue or purple fish that you might see in a pet shop. (See image inModule 35A.) These are transgenic zebrafish that express GFP, RFPor other fluorescent proteins to give their entire body the brightfluorescent color.

1a. When designing the transgene, the developers needed tochoose appropriate transcriptional regulatory elements to driveexpression of the transgene. Among the following regulatoryelements, which is the most likely to drive expression offluorescent protein in the Glofish?
a. A liver specific promoter that is upregulated only at thetime of liver differentiation
b. A skin specific enhancer that is only upregulated bytoxically low pH levels
c. A liver specific promoter that is constitutive
d. A muscle specific enhancer that is constitutive
e. A muscle specific enhancer that is upregulated only atthe time of muscle differentiation

1b. True or false?
Regarding the design of the transgene,no start methionine sequence is required at the 5' end of thefluorescent protein coding sequence.

1c. True or false?
Regarding the design of the transgene, a stopcodon is required at the 5' end of the fluorescent protein codingsequence, followed by a poly adenylation signal.

1d. To facilitate insertion of the transgene into the zebrafishgenome, it is cloned into a Tol2 transposon. Plasmid carrying thetransposon with transgene is injected into 2 cell embryos. Duringinjection, a second plasmid is also injected. What does the secondplasmid most likely contain?
a. A reporter gene that expresses lacZ in the presence offluorescent protein
b. A gene providing resistance to gancyclovir
c. A gene providing resistance to neomycin
d. A gene encoding Tol2 transposase under control of apromoter that is only expressed in adult tissues
e. A gene encoding Tol2 transposase under control of aconstitutive promoter.