Page 5 - K. Development and regeneration

A 3 0 0 B **

* ** 3 0 0 0

4 4 /p )
/p ) 4 s e G

ll lin e s e ra 4 G D 2 0 0 in g le ce ll lin e ra c ife 2 0 0 0

b le ce ife c to S u L e la tive to D 1 0 0 0

ta u L tive 1 0 0 (R
S la
e
(R
0
0 .3 μ g C trl + - -

0 6 .0 μ g B A C - + +
0 .3 μ g C trl + - - T o l2 tra n sp o sa se - - +

V e c to r 0 .6 6 7 μ g
6 .0 μ g B A C - + + Luciferase

T o l2 tra n sp o sa se

V e c to r 0 .6 6 7 μ g - - + Actin

C

0 .8
0 .3 μ g C trl
6 .0 μ g B A C
0 .6
B A C & T o l2
)
lls
e
μ C 0 .4
5
(E
0 .2

0 .0
3 6 9 1 2 1 5 1 8 2 1 2 4 2 7 3 0

D a y s

A B

3 0 0
1 0 0 ***
*

)
r 6 l
e 0 ** ve
e b m trl # *** e le trl) 2 0 0
BAC ll lin ce u n y C ll lin ce n tio C to

(200kb ~ 300kb) le g p o c to e tiv 5 0 le g crip tive la

Targeting in S e n e la e in S s n ra e (R 1 0 0

Vector G (R T ***

0 0

0 .3 μ g C trl + - - 0 .3 μ g C trl + - -

6 .0 μ g B A C - + + 6 .0 μ g B A C - + +

T o l2 tra n sp o sa se - - + T o l2 tra n sp o sa se - - +
Tol2 V e c to r 0 .6 6 7 μ g V e c to r 0 .6 6 7 μ g

CAM

Targeting

Vector

A B C

****

** * ****
6 0 0
1 5 0 0 ))
id ) .3 2 0 0
la sm trl(0
s e 4 0 0 trl(0 .3 )) s e
ra ** * s e ra C
c ife ra 1 0 0 0 ife to

u e la tive to P c ife **** c
L 2 0 0 u e la tiv e to C u L tiv e 1 0 0

(R L la e ****

(R 5 0 0 (R
0
C o n tro l 0 .3 μ g + - - 0
C o n tro l (μ g ) - - - - - -
B A C 6 .0 μ g - + +
B A C (μ g ) 8 8 8 8 8 8
T o l2 tra n s p o s a s e V e c to r - + - 0
C o n tro l (μ g ) 0 .3 - - - - - - B A C : T o l2 tra n s p o s a s e - 9 :0 .5 9 :1 9 :3 9 :6 9 :9
T o l2 tra n s p o s a s e m R N A - - + m R N A ra tio
B A C (μ g ) - 2 4 8 1 6 3 2 6 4

A B

3’ K CMV 5’ 5’ CMV K 3’
XmaI XhoI

3’ 5’
e1e2 e3 BAC targeting vector (BTV)
A Genome 3’arm 5’arm Genome
Reference

Conservation
5’ CMV K 3’

C BAC targeting cassette (BTC)

5’’ A SV40 N 3’’
C D e1e2 3’ K CMV 5’ e3

5’’ SV40 N A 3’’ CMV Tol2
BamHI XhoI

CAM targeting vector (CTV) N SV40 SV40 N A

E
5’’ SV40 N A 3’’
2
~
2
CAM targeting cassette (CTC) CMV SV40 N

Genome 5’arm 3’arm Genome
K A N 5’ 3’ 5’’ 3’’ Reference
Conservation
FRT SA SP Luciferase KanR AmpR NeoR Tol2 arm 5’ HR 3’ HR 5’’ HR 3’’ HR