Zhuet al.,Science 375 , eabg9765 (2022) 21 January 2022 4 of 11
B
D Time-lapse imaging reveals MultiFate-2.3 tristability
High TMP, Image at 119h 0h 119h
A-only
B-only
A + B
A Experimental MultiFate-2 design
37bs-37bs
inactive
complex
BCRbs-BCRbs
AD AD
AD
AD AD
AD
AD AD
AB
6x or 10x 6x or 10x
Dox
4-OHT
or Dox
C MultiFate-2 cell lines exhibit bistability and tristability
A-only
B-only A + B
OFF
OFF A-only
B-only A+B
OFF A-only
B-only A+B
+ AP1903
+ TMP
3 days
A-only
B-only A + B
OFF
+ AP1903
+ TMP
3 days
Unstable states - At least 10% cells escaped initial states
MultiFate-2.3
High TMP
Day 0 Day 18
OFF
A-only
A + B
B-only
Initial state
Type II tristability
Low TMP
Day 0 Day 18
Bistability
MultiFate-2.2
Day 0 Day 18
Low TMP
Day 0 Day 18
High TMP
Type II tristability Bistability
OFF
A-only
B-only
A + B
Initial state
MultiFate-2.1
Day 0 Day 18
Low TMP
Day 0 Day 18
High TMP
Type II tristability Bistability
OFF
A-only
B-only
A + B
Initial state
0
20
40
60
80
100
% of cells
60h
OFF A-only
A+B
TF A Expression
(mCherry) (a.u.)
102
103
104
105
106
102103104105106
TF B Expression (mCitrine) (a.u.)
FACS
Mixed at equal ratio
TF A Expression
(mCherry) (a.u.)
TF B Expression (mCitrine) (a.u.)^103
104
105
106
102 103 104 105 106
TF A Expression
(mCherry) (a.u.)
103
104
105
106
102 103 104 105 106
B-only
Fig. 3. MultiFate-2 generates multiple stable states.(A) The experimental
MultiFate-2 design uses two self-activation cassettes differing only in their
fluorescent proteins and their ZF DNA binding domains and binding sites. Each
cassette expresses FKBP-ZF-VP16-DHFR-IRES-FP-PEST, where ZF represents either
BCRZFR39A or 37ZFR2AR11AR39AR67A and FP represents either mCherry or
mCitrine, for A and B, respectively. Detailed construct maps and differences
amongMultiFate-2linesareshownintablesS2andS3.(B) MultiFate-2.1 cells
spontaneously activate A, B, or both cassettes upon addition of 100 nM AP1903
and 10mM TMP. Cell percentages in OFF, A-only, B-only, and A+B states were
quantified and plotted as a square with four colored circles ( 31 ); a.u., arbitrary
units. (C) Three MultiFate-2 lines all exhibited type II tristability in the high-TMP
condition and bistability in the low-TMP condition. In all conditions, we added
100 nM AP1903. Exact concentrations of TMP are shown in figs. S9 to S11. Unstable
states, defined by states having more than 10% cells escaping their initial states
after 18 days, were marked in pink rectangles. Each square represents the mean
fractions of three biological replicates. Initial A-only, B-only, and A+B cells were
sorted from a population of cells in different states, while initial OFF cells came from
cells in regular CHO media without any inducers. (D) A-only, B-only, and A+B states
were each stable during growth from single MultiFate-2.3 cells into colonies over
5 days (119 hours) under a time-lapse microscope. Left: We first sorted mixed
MultiFate-2.3 cell populations to separate cells in three different states. Then
we seeded cells in these three states at equal ratio in the same well and performed
time-lapse imaging ( 25 ). Scale bars, 500mm for the wide-field image, 100mm for
zoomed-in images.“High TMP”= 100 nM AP1903 + 10mM TMP.
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