Page 3 - K. Development and regeneration

A B C 4 Fer1L4

Fer1L4 is not targeted by LP

200 bp 3
Relative Copy No. 2 ▼ ▼ ▼
Fer1L4 Fwd Fer1L4 Rev

Fer1L4 1 1 2 3 9

DIC mCherry Merged Fer1L4 is targeted by LP 1 ▼ ▼ ▼ ▼ ▼

3,300 bp

Fer1L4 Fwd CMV Fer1L4 Rev 0
1 mCherry TK 1 2 3 9

Lox5171 LoxP

Primer Sequence (5' to 3') Primer Sequence (5' to 3') Negative control mCherry positive #01 mCherry positive #02 mCherry positive #03 mCherry positive #04 mCherry positive #05 mCherry positive #06 mCherry positive #07 mCherry positive #08 mCherry positive #09 mCherry positive #10 mCherry positive #11 mCherry positive #12
Fer1L4 GCACTGCGCCATGGCTCT Fer1L4 GACGGGTTATAGAGCTGG
Fwd Rev
GAPDH GCACCACCAACTGCTTAG GAPDH AGTCTTCTGGGTGGCAGT
Fwd C Rev GA

D E

rd
1 590 bp 1 st 590 bp 2 nd 436 bp 3 348 bp
st
2 nd 436 bp
C
3 348 bp negative control #4 #6 #8 #9 #11 negative control #4 #6 #8 #9 #11 negative control #4 #6 #8 #9 #11
rd
CMV
1 mCherry TK 1 2 3 9 600

Lox5171 LoxP 500
Primer Sequence (5' to 3') Primer Sequence (5' to 3') 300
st
st
1 Fwd TCACGGGCTAGCAAGGAC 1 Rev GAGCCGTACATGAACTGA
2 nd Fwd TACCCGATCCCAGCTC 2 nd Rev CCCTCGATCTCGAACT
rd
rd
3 Fwd CGACGGTATGGGAAGA 3 Rev CGTGGCCGTTCACGGAGC
F
Genome 5’ Homologous arm 5’ Homologous arm Lox5171

A C Fer1L4 1 2 3 9 D

Fer1L4
Fer1L4 1 2 3 9 Cleavage CMV
1 mCherry TK 1 2 3 9
AGACGCCTAACAGAGCTGCCAGGCA
TCTGCGGATTGTCTCGACGGTCCGT Lox5171 LoxP
5’AGACGCCTAACAGAGCTGCCAGG
Cas9 gRNA 3’ CRE CRE
CMV
mCherry TK SV40
Lox5171 LoxP
5′ HA 3′ HA eGFP Neo
Lox5171 LoxP
Landing pad (LP) homologous recombination

CMV Targeting vector
mCherry TK
Lox5171 LoxP
B 5’ homologous arm 3’ homologous arm

SV40
RMCE Landing pad Fer1L4 SV40
eGFP Neo
1 eGFP Neo 1 2 3 9
Lox5171 LoxP
Lox5171 LoxP
Targeting vector (TV) CMV
1 mCherry TK 1 2 3 9

Lox5171 LoxP

A B 332 bp C

SV40 5
1 eGFP Neo 1 2 3 9 4
Lox5171 LoxP **
Primer Sequence (5' to 3') Primer Sequence (5' to 3') 3
Fwd AGACTGAGCCACAGCACTGC Rev CGCAGCCCTCCATGGTGAAC Percentage of cells expressing GFP, but not mCherry (%) 2

Before RMCE After RMCE 0
DIC mCherry 1

Before RMCE After RMCE

GFP Marged 500 D E 82
300
Percentage of cells expressing GFP, but not mCherry (%) 3 Percentage of cells expressing GFP, but not mCherry (%) 79

Save cost & time! 5’ Homologous arm Lox5171 GFP 5 4 ** 81 **
80

6
Decrease 2 4 **
Minimum 12 months processing time 1 2

Save cost & time! 0 g CRE g CRE g CRE g CRE 0

    Before RMCE Before sorting After sorting
0.5 1.0 2.0 4.0

2 .0  g Ta rg e tin g ve c to r
After
RMCE