
Supplementary Table 1: List of the DNA constructs used in this. The constructs
and the complete sequence information for each construct are available upon
request.
Construct name
A422.
RacFRET-3’nos
B441.
Venus-3’globin
B757.
YPet-3’globin
B758.
SECFP-3’globin
B759.
CyPet-3’globin
Description
RacFRET biosensor without the C-terminal targeting
sequence; for expression in primordial germ cells (PGCs) in
zebrafish. Previously described by Kardash et al (20).
Experimental construct for measuring Rac activation pattern
in PGCs.
Venus for global expression. The open reading frame
encoding for the Venus protein (described in reference 15)
was cloned upstream of the globin 3’UTR. Acceptor control
construct used in the “Non-linked” control in combination
with the CFP variant.
YPet for global expression. The open reading frame
encoding for the YPet protein (described in reference 31)
was cloned upstream of the globin 3’UTR. Acceptor control
construct used in the “Non-linked” control in combination
with the CFP variant.
SECFP for global expression. The open reading frame
encoding for the SECFP protein (described in reference 15)
was cloned upstream of the globin 3’UTR. Donor control
construct used in the “Non-linked” control in combination
with the YFP variant.
CyPet for global expression. The open reading frame
encoding for the CyPet protein (described in reference 31)
was cloned upstream of the globin 3’UTR. Donor control
construct used in the “Non-linked” control in combination
with the YFP variant.
Description
Amount
injected
(pg)
360
150
150
150
150
Amount
Construct name
B760.
Venus-SECFP- 3’globin
B761.
YPet-SECFP-3’globin
B762.
YPet-CyPet-3’globin
B763.
RacFRET-NoCT-3’globin
B782.
RacFRET-zRac1CT3’globin
Construct name
injected
(pg)
Venus-SECFP tandem (Linked) for global expression.
Used to measure maximum YFP/CFP ratio. Venus and
SECFP proteins are separated by 13 amino acid linker:
LESGGGTGGGRGR. Control construct used in the
“Linked” control.
YPet-SECFP tandem (Linked) for global expression.
Used to measure maximum YFP/CFP ratio. YPet and
SECFP proteins are separated by 13 amino acid linker:
LESGGGTGGGRGR. Control construct used in the
“Linked” control.
YPet-CyPet tandem (Linked) for global expression.
Used to measure maximum YFP/CFP ratio. YPet and
CyPet proteins are separated by 13 amino acid linker:
LESGGGTGGGRGR. Control construct used in the
“Linked” control.
RacFRET biosensor without C-terminal targeting
sequence for global expression. The 3’ nanos UTR in
RacFRET (described by Kardash et al (20)) was
replaced by the globin 3’ UTR. Experimental construct
for measuring Rac activation pattern in somatic cells in
zebrafish. The subcellular localization of a biosensor is
cytosolic.
RacFRET biosensor with the zebrafish Rac1 C-terminal
targeting sequence for global expression. In RacFRET
(B763), a stop codon after SECFP was eliminated and
the C-terminal domain of zebrafish Rac 1 (amino acids:
172-192) was introduced upstream of the globin 3’UTR.
Experimental construct for measuring Rac activation
pattern in somatic cells in zebrafish. This biosensor is
localized mainly to the nucleus, and also detected in the
cytosol and at the membrane.
Description
300
300
300
300
300
Amount
injected
(pg)
B783.
RacFRET-zRac1CTNoNLS-3’globin
B784.
zRac1N17-3’Globin
B786.
zTiam1-DhPh-3’globin
B787.
RacV12-FRET-3’globin.
B800.
mCherry-zRac1-3’globin
RacFRET biosensor lacking the nuclear localization
signal (NLS) for global expression. The NLS in Rac1 Cterminus was eliminated (described by Kardash et al
(20)). Experimental construct for measuring Rac
activation pattern in somatic cells in zebrafish. This
biosensor is localized to the cytosol and to the
membrane.
Dominant-negative Rac1 (described by Kardash et al
(20)) for global expression. Dominant-negative Rac1
construct for measuring the low level of Rac activity in
the RacFRET biosensor.
DH-PH active domain from zebrafish Tiam1 (described
by Kardash et al (20)) for global expression. The open
reading frame of the DH-PH domain of Tiam1 was fused
to the globin 3’UTR. This construct is used as a control
to activate RacFRET biosensor.
A positive control for cytosolic RacFRET biosensor for
global expression. The G12V substitution was
introduced into the coding region of Rac within the
biosensor as previously described by Kardash et al (20).
Used to measure high levels of Rac activation in
somatic cells.
mCherry-zebrafish Rac1 fusion for global expression.
Zebrafish Rac1 (BC071548) was fused to mCherry and
cloned upstream of the globin 3’UTR. This construct is
used to follow Rac1 subsellular localization in somatic
cells.
300
300
300
300
300