The Myc-tagged humanKRASG12Vwas ampli-
fied frompBABE-KRASG12V-puro (Addgene,
#9052)andinsertedtotheMCS.Theresultant
pBS31-CAG-L-KRASG12V-WPRE-pAplasmid was
co-electroporated with an FLP expression plas-
mid into KH2 ES cells and selected clones with
Cre-inducibleKRASG12Vexpression were used
to generateL-KRASG12Vmice according to stan-
dard procedures. The same strategy was used to
generateL-p21mice orL-p16mice using Myc-
Flag-tagged cDNAs for mousep21or mouse
p16obtained from Origene (#MR227529 or
#MR227284, respectively). Obtained founder
mice (C57BL/6×129Sv background) were back-
crossed to C57BL/6 at least twice before use
in breeding for experimentation. To generate
iL-p21transgenic mice on a pure C57BL/6
background, the following targeting construct
was cloned:pTRE2-LoxP-STOP-LoxP(LSL)-p21-
Myc-Flag-WPRE-pAusing theTRE2promoter
andLSLfrom theAi139transgene (Addgene,
#114426) andp21-Myc-Flagfrom theL-p21
transgene (Origene, #MR227529) as described
above. Homology arms spanning 968 bp at the
5 ′-end and 937 bp at the 3′-end flanked by
sgRNA target sites were used to target the con-
struct into theCol1a1locus of C57BL/6NHsd
(Envigo) zygotes using CRISPR-Cas9-mediated
gene editing withCas9mRNA (Trilink Bio-
technologies, #L-7606) andCol1a1-specific
sgRNA 5′-GAGGTTCATGAGCCCTCAAA-3′.
Obtained founder mice were backcrossed to
C57BL/6 once before use for experimentation.
To generatep21floxedmice on a pure C57BL/6
background, a targeting vector containingp21
exon 2 flanked by LoxP sites and homology
arms spanning 861 bp at the 5′-end and 819 bp
at the 3′-end flanked by sgRNA target sites
(5′sgRNA: 5′-TCTTGGTGATTAACTCCATC-3′
and 3′sgRNA: 5′-CCATAGGCGTGGGACCTCGT-
3 ′) was cloned. The resultant targeting vector
was used to target the construct into thep21
locus of C57BL/6NHsd (Envigo) zygotes using
CRISPR-Cas9-mediated gene editing withCas9
mRNA (Trilink Biotechnologies, #L-7606) ( 40 ).
Obtained founder mice were crossed to C57BL/6
at least once before use for experimentation.
Rbfloxedmice (#026563),Ai14transgenic ani-
mals (#007914), andAi139transgenic mice
(#030219) were purchased from The Jackson
Laboratory. The following cohorts were gen-
erated for experimentation in this study:
Ai14/+andAi14/+ L-KRASG12V/+(C57BL/
6×129SV mixed background; fig. S2),Ai14/+
andAi14/+ L-p21/+(C57BL/6×129SV mixed
background; Figs. 3 and 4, figs. S13 and S14),
Ai14/+andAi14/+ L-p16/+(C57BL/6×129SV
mixed background; fig. S16),Ai14/+and
Ai14/+ L-KRASG12V/+andAi14/+ L-KRASG12V/+
p21floxed/floxedandAi14/+ L-KRASG12V/+Rbfloxed/floxed(C57BL/6×129SV mixed back-
ground; Figs. 5 and 6 and fig. S18),Ai139/+and
Ai139/+ iL-p21/+(C57BL/6 pure background;
Fig. 8 and fig. S20). Mice were aged until 4 to
6 months of age before use for experimentation
unless otherwise noted. Experimental proce-
dures involving laboratory mice were reviewed
and approved by the Institutional Animal Care
and Use Committee of the Mayo Clinic.Cellculture
Mouse embryonic fibroblasts (MEFs) were gen-
erated and cultured as described previously
with each line being derived from a separate
C57BL/6 E13.5 embryo containingINK-ATTAC
( 12 , 41 ). These lines were expanded at 3% oxy-
gen and used for experiments between pas-
sage (P)3 and P6. IMR-90 cells were purchased
from ATCC (#CCL-186) at P10 and cultured in
the same medium as used for MEFs. IMR-90
cells were used for experimentation between
P14 and P18. Human dermal fibroblasts (HDFs)
were generated from human foreskin of young,
healthy donors (2 days to 13 years of age) by the
Biochemical Genetics Laboratory at the Mayo
Clinic. Each line was derived from a separate
donor. HDFs were cultured in the same me-
dium as used for MEFs and used for experi-
mentation between P5 and P8. HUVECs were
purchased from ATCC (#PCS-100-013) and wereSturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 8 of 15
p212dON
EV2dON4dOFF
EV p21A CM EV (2dON)
CM p21-OE (2dON)
CM EV (2dON 4dOFF)
CM p21-OE (2dON 4dOFF)Bd0 d2 d4 d6 d8CM 2dON 4dOFF+doxMigrating macrophages (fold)***n=6n=6 n=6n=6ns2dON 4dOFF2dONCM 2dON–doxp21PonSp21-Myc-FlagEV (2dON) p21-OE (2dON)
EV (2dON 4dOFF) p21-OE (2dON 4dOFF)CDCxcl14 Igfbp3 Gas6 Loxns***
nsns*ns** **Appns*n=9n=9 n=9n=9Relative expression00.51.01.52.02.500.51.01.52.02.500.51.01.52.02.500.51.01.52.000.51.01.52.00123Fig. 7. Cells normalizing p21 cease to produce a PASP and are released from immunosurveillance.(A) Schematic overview of CM preparations from dox-inducible
p21-OE MEFs. (B) Representative immunoblot for p21. PonS served as loading control. (C) Transwell macrophage migration with CM from indicated MEFs. (D) RT-qPCR
of the indicated MEFs. Data represent means ± SEM. The blot shown in (B) is representative of two independent experiments, and data shown in (C) and (D) were
pooled from two and three experiments, respectively. ns, not significant. P< 0.05; P< 0.01; P< 0.001. Two-way ANOVA with SidakÕs correction [(C) and (D)].
RESEARCH | RESEARCH ARTICLE