ア ニマルキャップ細胞群を用いた 胚全体構造の構...
Transcript of ア ニマルキャップ細胞群を用いた 胚全体構造の構...
ニマルキャップ細胞群を用いた胚全体構造の構築への挑戦
SFC-SWP 2015-010
AUTUMN2015年度 秋学期
ア
研究会優秀論文
慶應義塾大学湘南藤沢学会
森本健太 環境情報学部 4年
先端生命科学研究会
2015
Attempt to create overall embryonic structure
by the combination of artificially induced blastula centers
2
(AC)
RNA-seq Chordin Sizzled
X mRNA
gain-of-functional
CRISPR/Cas
β-Catenin VegT Siamois Twin
AC
AC
BCNE Nieuwkoop
Wnt8 mRNA VegT mRNA AC
AC AC
Keywords: , , RNA-seq, CRISPR/Cas,
3
...........................................................................................................................2
...................................................................................................................................3
1 .......................................................................................................................5
1.1. ...............................................................................................5
1.2. ...............................................................7
1.3. .......................................................9
2 RNA-seq ......................................12
2.1. .....................................................................................................................12
2.2. .....................................................................................................................15
2.2.1. ..........................................................................15
2.2.2. RNA cDNA ................................................................................15
2.2.3. RNA-seq ........................................................15
2.2.4. pCS2 ..................................................16
2.2.5. mRNA ................................................................................................16
2.3. .....................................................................................................................16
2.3.1. RNA-seq ....................16
2.3.2. SP-X ......................................19
2.4. .....................................................................................................................21
2.4.1. RNA-seq ............................................21
2.4.2. SP-X .................................................................22
3 RNA CRISPR/Cas ....24
3.1. .....................................................................................................................24
3.2. .....................................................................................................................26
3.2.1. ..........................................................................26
3.2.2. gRNA PAMmers gRNA ..................................................27
4
3.2.3. Cas9-nonNLS mRNA ........................................................................29
3.3. .....................................................................................................................29
3.3.1. CRISPR/Cas .....................................29
3.2.2. CRISPR/Cas .................31
3.4. .....................................................................................................................32
3.4.1. CRISPR/Cas ..................32
3.4.2. CRISPR/Cas ..................................34
4 .................36
4.1. .....................................................................................................................36
4.2. .....................................................................................................................38
4.2.1. ..........................................................................38
4.2.2. B-N-AC .....................................................................39
4.2.3. HE ..........................................................................................................40
4.2.4. BDA .......................................................................................................41
4.3. .....................................................................................................................41
4.3.1. AC ..............................................41
4.3.2. HE BDA ...............................42
4.4. .....................................................................................................................45
4.4.1. ..................45
4.4.2. ..........................................46
5 .....................................................................................................................48
.................................................................................................................................50
.........................................................................................................................53
.................................................................................................................................58
5
1
2
19
17
2
( Cell)
17~18
1)
2) 3)
1.1.
6
Trim36 (tripartite motif containing 36) Dnd1 (DND microRNA-mediated
repression inhibitor 1)
(Cuykendall et al., 2009) Dnd1 RNA
Dnd1 trim36 mRNA 3’-UTR
Trim36
(Mei et al., 2013) Trim36 E3
Dnd1 Trim36
30
30
(Houliston and Elinson, 1991) 4
(Kikkawa et al., 1996)( 1) Wnt/β-Catenin
β-Catenin β-Catenin TCF/LEF
Siamois Twin
(Molenaar et al., 1996; Brannon et al., 1997) Wnt/β-Catenin
Wnt11 Wnt8
(Tao et
al., 2005)
7
1
30
1.2.
(SpO)
SpO
(Nieuwkoop, 1969)
8
Nieuwkoop
(Nieuwkoop, 1973) Nieuwkoop SpO
4 T VegT
β-Catenin Wnt/β-Catenin
(Zhang and King, 1996) VegT
Wnt/β-Catenin Nieuwkoop
(Clements et al., 1999) VegT
Wnt/β-Catenin
Wnt/β-Catenin Siamois
Twin Siamois Twin BMP Chordin Noggin
Nieuwkoop BCNE (blastula Chordin- and
Noggin-expressing) ( 2) BCNE
(Kuroda et al., 2004) Nieuwkoop
Nodal BCNE
SpO Nieuwkoop Nodal
BCNE
(Agius et al., 2000; De Robertis and Kuroda, 2004)
9
2
(Stage 8) β-Catenin VegTNieuwkoop β-Catenin
BCNE SpO (Stage 10) BCNENieuwkoop Nodal
1.3.
FGF(Fibroblast growth factor)
(AC) AC
AC FGF
(Slack et al., 1989) FGF
(Kinoshita and
Asashima, 1995)( 3) SpO
10
Nodal TGF-β Nodal
Nieuwkoop
(Agius et al., 2000; De Robertis and Kuroda, 2004)
AC
(Ariizumi and Asashima, 2001)
3
(Stage 9) ACAC
11
RNA-seq
Chordin Noggin
Vent1 Sizzled
X Trim29 3
X
CRISPR/Cas
β-Catenin VegT mRNA Siamois Twin
mRNA CRISPR/Cas
AC
AC BCNE Nieuwkoop
12
2 RNA-seq
2.1.�
4
(Kikkawa et al., 1996)
(SpO)
SpO BCNE (blastula Chordin- and Noggin-expressing)
Nieuwkoop Nodal
(Agius et al., 2000; De Robertis and Kuroda, 2004) BCNE
Siamois Twin
BMP Chordin Noggin
(De Robertis and Kuroda, 2004; Kuroda et al., 2004) Siamois Twin
BMP4 BMP
(Ishibashi et al., 2008) β-Catenin VegT mRNA
Nieuwkoop Nodal
( 4) 20
13
(De Robertis and Kuroda, 2004)
(Tompsett et
al., 2013; Rotman et al., 2013)
RNA-seq
( SP-X)
Xolloid
Xolloid BMP Chordin C1
C2 C3 C4 Chordin
BCNE Xolloid Chordin
BMP
(Larraín et al., 2001; Oelgeschläger et al., 2000) SP-X
xHtrA1
xHtrA1
Biglycan Syndecan-4 Glypican-4
FGF (Fibroblast growth factor) GAG
(glycosaminoglycan) FGF-GAG FGF
FGF (Hou et al., 2007)
SP-X
SP-X SP-X
gain-of-functional SP-X
pCS2 pCS2-SP-X
14
SP-X mRNA
4
(Stage 10)BMP BMP
Chordin Noggin
15
2.2.
2.2.1.
�
(HCG10000, )
600 U
0.1x
(0.1x SS: 1x SS [58 mM NaCl, 0.67 mM KCl, 3.4 mM CaNO3-4H2O, 0.83
mM MgSO4-7H2O, 0.1 g/l Kanamycin Sulfate, 5 mM Tris-HCl, pH 7.4] 10
) 22 ˚C 1%
(100 ml 0.1x 1 ml
[#208-01026, ] 5N NaOH 3 ml )
2.2.2. RNA cDNA
3 15
RNA RNA ( , pH 5 76 ml Guanidine
Thiocyanate 23.6 g Ammonium Thiocyanate 15.2 g 3 M Sodium Acetate, pH 5.3
33.4 ml (#072-04945, ) 10 ml Nuclease-free
water (#129114, QIAGEN) 50 ml
RNA 15 µl Nuclease-free water
RNA (#48190-011, life
technology) RNase inhibitor (#SIN-201, ) dNTPs (#201913, QIAGEN)
M-MLV, RNaseH+ (#187-01286, Wako) 30 ˚C 10
42 ˚C 60 72 ˚C 10 cDNA
2.2.3. RNA-seq
Stage 10 (n=15)
RNA illumina HiSeq 100 bp Paired end
16
RNA-Seq Xenbase
(http://www.xenbase.org) Xenopus laevis (JGI 7.0)
TopHat ver. 2.0.9 (Trapnell et al., 2009)
Cufflinks ver.2.1.1 (Trapnell et al., 2010)
2.2.4. pCS2
pUC57-SP-X EcoRI XhoI
SP-X DNA
pCS2 EcoRI XhoI Antarctic
Phosphatase (#M0289L, NEB) pCS2
SP-X TaKaRa Ligation Kit Ver. 2 (#6022, TaKaRa)
2.2.5. mRNA
pCS2 SP-X pCS2-SP-X 5 ng Asp718
15 µl Nuclease-free water mRNA
6 µl SP6 mMessage mMachine Kit (#AM1340, Ambion)
37 ˚C 2 DNase 37 ˚C 15
mRNA
20 µl Nuclease-free water 500 ng/µl
2.3.
2.3.1. RNA-seq
Stage 10
RNA-seq
RNA-seq
45,336,894 42,035,856
17
TopHat
ver. 2.0.9 Xenopus laevis (JGI 7.0)
86.75%
86.95% Cufflinks ver.2.1.1 cuffdiff
2
Stage 10 19896
143
43
SP-X Trim29 3 (USP75AA USP107AA
USP276AA )
5
18
5
(A) X YFPKM(fragments per kilobase of exon per million mapped fragments)cuffdiff (B)
FPKM
BMP
19
2.3.2. SP-X
SP-X RNA-seq
SP-X SP-X gain-of-functional
SP-X
SP-X mRNA 4 2 4 nl 800
pg ( 6A) SP-X mRNA
28.8% (13/45) 84.4% (38/45)
( 6D, E, H, I, J, K)
(
6B) 13.3% (6/45) 15.6% (7/45)
( 6I, K)
( 1, 2) SP-X mRNA
20
6 SP-X mRNA
(A) 4 SP-X mRNA 4 24 nl mRNA 800 pg (B)
(C-E) Stage 29 C D ED 2 28.8% (13/45)E 2 84.4% (38/45)
(F-K) Stage 46 F G H-K H I 213.3% (6/45) J K 2
84.4% (38/45)1 mm
21
2.4.
RNA-seq
SP-X
1) RNA-seq
2) SP-X
2.4.1. RNA-seq
Stage 10 RNA RNA-seq
Chordin Noggin ADMP
Sizzled Bambi Wnt8
BMP4
BMP Stage 10
BMP4 Stage 8 Stage 12
Stage 10
X
Trim29
(tripartite motif containing 29) Trim29 MYST
22
Tip60 p53
(Sho et al., 2011; Yuan et al., 2010)
p53 Trim29
(Cordenonsi et al., 2003)
75
107 276 3
32 ELABELA
(Chng et al., 2013)
3
2.4.2. SP-X
SP-X
4
SP-X mRNA
( ; 13/45 ; 38/45 6D, E, H, I, J, K)
Chordin Noggin Follistatin BMP
BMP
(Piccolo et al., 1996; Zimmerman et al., 1997) BMP
SP-X mRNA
mRNA
MO
SP-X
( 4H, J) Pax6
23
Otx2 (Kenyon et al., 1999)
Pax6
(Chiang et al., 1996; Macdonald et al., 1995)
(Strähle et
al., 1993) SP-X mRNA Pax6
(Tran et al., 2007; Hoff et al., 2013; Horb et al., 1999) SP-X
mRNA
SP-X
24
3 RNA CRISPR/Cas
3.1.
20
gain- or loss-of-functional
(MO)
Zinc Finger TALEN CRISPR/Cas
F0 (Kim et al.,
1996; Cermak et al., 2011; Garneau et al., 2010) 4
Cas9 RNA
gRNA RNA PAM-presenting
oligonucleotides (PAMmer) 3 RNA
(O’Connell et al., 2014) ( 7)
CRISPR/Cas RNA
MO
MO
25
CRISPR/Cas siRNA
RNA
mRNA
CRISPR/Cas
CRISPR/Cas
β-Catenin VegT 2
Siamois Twin
β-Catenin
Wnt/β-Catenin
T-box VegT
β-Catenin VegT Nieuwkoop
β-Catenin VegT
BCNE (blastula Chordin- and Noggin-expressing)
BCNE
Siamois Twin BMP Chordin
Noggin (De Robertis and Kuroda, 2004; Kuroda et al., 2004)
Siamois Twin BMP4 BMP
(Ishibashi et al.,
2008) Nieuwkoop Nodal
MO
β-Catenin 2
(Heasman et al.,
2000) VegT Nieuwkoop
VegT (Heasman et al., 2001)
Siamois Twin BMP
26
(Ishibashi et al., 2008)
CRISPR/Cas β-Catenin
VegT Siamois Twin gRNA PAMmers
Cas9-nonNLS mRNA gRNA PAMmer 3
7 CRISPR/Cas
(A) CRISPR/Cas PAMgRNA Cas9 ( )
(B) CRISPR/Cas RNA mRNAPAM DNA PAMmer gRNA
Cas9 RNA
3.2.
3.2.1.
�
(HCG10000, )
600 U
0.1x
(0.1x SS: 1x SS [58 mM NaCl, 0.67 mM KCl, 3.4 mM CaNO3-4H2O, 0.83
mM MgSO4-7H2O, 0.1 g/l Kanamycin Sulfate, 5 mM Tris-HCl, pH 7.4] 10
) 22 ˚C 1%
(100 ml 0.1x 1 ml
27
[#208-01026, ] 5N NaOH 3 ml )
3.2.2. gRNA PAMmers gRNA
gRNA 20
PAMmer 19 NCBI RefSeq
4 PAMmer
( 8A) gRNA 2
gRNA DNA RNA polymerase
in vitro T7
gRNA FW (60 b) Cas9
gRNA RV (76 b) 2 gRNA
(100 µM each) 1 µl 10x PCR buffer for KOD-Plus-Neo 10 µl dNTPs (2
mM each) 1 µl 25 mM MgSO4 3 µl KOD-Plus-Neo (#436300, ) 1 µl
32 µl 94 ˚C 30 55 ˚C 1 68 ˚C 30
gRNA DNA gRNA DNA
500 ng DNA T7 RNA
polymerase (#10881767001, Roche) 37 ˚C 1
37 ˚C 30 DNase
gRNA Nuclease-free water
PAMmer 8B Integrated DNA
Technologies (IDT )
28
8 gRNA PAMmer
(A) gRNA PAMmer (B) gRNA-FWgRNA-RV PAMmer
29
3.2.3. Cas9-nonNLS mRNA
pCS2 NLS Cas9
(Cas9-nonNLS) pCS2-Cas9-nonNLS 5 ng Asp718
15 µl Nuclease-free water mRNA
6 µl mMESSAGE mMACHINE SP6 Transcription
Kit (#AM1340, Ambion) 37 ˚C 2 DNase
37 ˚C 15
mRNA 20 µl Nuclease-free water
1000 ng/µl
3.3.
3.3.1. CRISPR/Cas
CRISPR/Cas
β-Catenin VegT 2 1 β-Catenin gRNA
100 pg β-Catenin PAMmer 20 pg Cas9-nonNLS mRNA 1000 pg
β-Catenin
10.3% (9/87) 37.9% (33/87)
20.7% (18/87)
( 9E-G) CRISPR/Cas
Cas9 β-Catenin gRNA 100 pg β-Catenin PAMmer 20 pg
10.5% (6/57) 19.2%
(11/57) ( 9C,
D, G)
30
9 CRISPR/Cas β-Catenin
(A) 1 (B) C-F(C, D) β-Catenin gRNA 100 pg β-Catenin PAMmer 20 pg C
(36/57) D (11/57) (E, F) Cas9-nonNLS mRNA 1000 pg β-Catenin gRNA 100 pg β-Catenin PAMmer 20 pg
(KD) E (9/87) F(33/87) (G) β-Catenin
68.9% Typical Mildexo 1 mm
VegT VegT gRNA 100 pg VegT PAMmer 20 pg
Cas9-nonNLS mRNA 1000 pg 1 15.4%
(12/78) 15.4% (12/78)
14.1% (11/78) β-Catenin
( 10E-G) VegT gRNA 100
pg VegT PAMmer 20 pg 14.3% (9/63)
19.0% (12/63) ( 10C, D,
G)
31
10 CRISPR/Cas VegT
(A) 1 (B) C-F(C, D) VegT gRNA 100 pg VegT PAMmer 20 pg C
(33/63) D (12/63) (E, F) Cas9-nonNLS mRNA1000 pg VegT gRNA 100 pg VegT PAMmer 20 pg (KD)
E (12/78) F (12/78) (G) VegT 46.2%
Typical Mild exo1 mm
3.3.2. CRISPR/Cas
Siamois Twin
CRISPR/Cas 8
1 Siamois gRNA 50 pg Twin gRNA 50 pg Siamois PAMmer
10 pg Twin PAMmer 10 pg Cas9-nonNLS mRNA 1000 pg
9.7% (6/62) 45.2%
(28/62) 12.9% (8/62)
( 11E-G) Siamois gRNA 50 pg Twin gRNA 50pg Siamois
PAMmer 10 pg Twin PAMmer 10 pg 15.4% (8/52)
7.7% (4/52) (
11C, D, G)
32
11 CRISPR/Cas Siamois/Twin
(A) 8 2 (B) C-F(C, D) Siamois gRNA 50 pg Twin gRNA 50 pg Siamois PAMmer 10 pg
Twin PAMmer 10 pg C (36/52) D(4/52) (E, F) Cas9-nonNLS mRNA 1000 pg Siamois gRNA
50 pg Twin gRNA 50 pg Siamois PAMmer 10 pg Twin PAMmer 10 pg(KD) E (6/62) F
(28/62) (G) Siamois/Twin 67.7%Typical Mild
exo 1 mm
3.4.
CRISPR/Cas
β-Catenin VegT
Siamois/Twin β-Catenin VegT
Siamois/Twin MO
1) CRISPR/Cas
2)
3.4.1. CRISPR/Cas
CRISPR/Cas β-Catenin 10.3% (9/87)
VegT 15.4% (12/78)
33
Siamois/Twin 9.7% (6/62)
CRISPR/Cas
MO 70~80%
DNA PAMmer
PAMmer DNA
locked nucleic acids 2'-OMe
phosphorothioate
CRISPR/Cas RNA
(O’Connell et al., 2014)
phosphorothioate DNA DNA
PAMmer phosphorothioate
(Woolf et al., 1990; Hulstrand et al., 2010)
10 DNA 5' 3' 5 2'-OMe
RNA 20 DNA/RNA (ASOs: antisence oligo
nucleotides)
(Pauli et al., 2015) RNA ASOs RNase H
DNA/RNA RNA ( 12A)
ASOs
β-Catenin ASOs
( 12C) CRISPR/Cas
ASOs
gRNA
CRISPR/Cas gRNA
34
gRNA
3’ GG 2 3 PAM
(Farboud and
Mayer 2015) gRNA RNA
12 DNA/RNA (ASOs) β-Catenin
(A) ASOs ASOs 5' 5 2'-OMe RNA 10 DNA3' 5 2'-OMe RNA(mN ) phosphorothioate (*
) RNA ASOs DNA/RNA RNase(B) Stage 36 (C) 1~2 β-Catenin-ASOs 300 pg
76.6%(92/120)
3.4.2. CRISPR/Cas
CRISPR/Cas RNA
MO
miRNA non-coding RNA
35
(Michiue and Asashima 2005) Cas9
gRNA PAMmer
Cas9
dCas9 GFP mRNA /
RNA- 1
mRNA (Chen et al., 2013)
36
4
4.1.
1
ES (embryonic stem cell) iPS (induced
pluripotent stem cell) (Yamanaka, 2008; Okita et
al., 2008)
(Gokhale and Andrews, 2008)
37
(AC)
ES
iPS AC
AC
AC AC
(Ariizumi and
Asashima, 2001) AC canonical Wnt
(Harland, 2000)
AC
AC
(SpO) . SpO
SpO SpO
(Spemann and Mangold, 1924) SpO BCNE
(blastula Chordin- and Noggin-expressing) Nieuwkoop
Nodal (Agius et
al., 2000; De Robertis and Kuroda, 2004)
SpO SpO BCNE Nieuwkoop����
�
(��� ) AC
AC
β-Catenin Wnt8/β-Catenin
(Christian and Moon,
1993) Wnt8 mRNA BCNE AC
38
Wnt8 mRNA VegT mRNA Nieuwkoop
AC (Ishibashi et al., 2008) 8 1
Wnt8 mRNA Wnt8 mRNA VegT
mRNA AC BCNE Nieuwkoop
AC
B-N-AC (BCNE and Nieuwkoop centers-contained AC)
( 13)
13
AC BCNE NieuwkoopAC
4.2.
4.2.1.
�
(HCG10000, )
600 U
0.1x
39
(0.1x SS: 1x SS [58 mM NaCl, 0.67 mM KCl, 3.4 mM CaNO3-4H2O, 0.83
mM MgSO4-7H2O, 0.1 g/l Kanamycin Sulfate, 5 mM Tris-HCl, pH 7.4] 10
) 22 ˚C 1%
(100 ml 0.1x 1 ml
[#208-01026, ] 5N NaOH 3 ml )
4.2.2. B-N-AC
8 1 Wnt8 mRNA 1 pg 1
VegT mRNA 160 pg ( 14A) 1x
1 0.1x Stage
9 Stage 9 1x
0.3 mm AC BCNE Nieuwkoop
B-N-AC B-N-AC
AC AC
( 14B) poly-HEMA
1x 22 ̊C poly-HEMA
40 mm (#1-8549-01, ) 100 %
EtOH 4% poly-HEMA (12% of 2-hydroxyethyl methacrylate
#18894-100, Polysciences) 500 µl 4% poly-HEMA
40
14 AC
(A) 8 1 Wnt8 mRNAWnt8 mRNA VegT mRNA (Stage 9) AC
(B) A
4.2.3. HE
PBSFA (10x PBS
1:1:8 ) 70% EtOH
100% EtOH
8 µm
100%
EtOH 70% EtOH 1
41
1 70% EtOH 100% EtOH
(038-01045, ) 1/10
4.2.4. BDA
HE 100% EtOH 70%
EtOH 1x wash buffer
(1 M Tris-HCl 1.5 M NaCl pH 7.5 10 ) Streptavidin-AP
solution (1x wash buffer 150 ml, Streptavidin-AP 7.5 µl) 3
1x wash buffer AP buffer (1 M Tris-HCl 50 ml, 0.5 M MgCl2 50
ml, DW 400 ml) 5 7 ml BM purple 140 ml AP buffer
4˚C 1
4.3.
4.3.1. AC
BCNE Nieuwkoop
B-N-AC
8 1 Wnt8 mRNA 1 pg
Wnt8 mRNA 1 pg VegT mRNA 160 pg
B-N-AC AC
(n = 138)
2 AC ( 15A, n = 30)
29.7% (41/138)
70.2% (97/138) 20
( 15B) 72
( 15C, D, 3)
42
15 B-N-AC AC
(A) AC (B) B-N-ACAC 32
(C, D) B-N-AC AC 72 CD ( ) cg, cement gland 1
mm
4.3.2. HE BDA
72 HE
Wnt8
mRNA Wnt8 mRNA VegT mRNA
BDA (biotin dextran-amine)
BCNE
Nieuwkoop
HE
( 16B-F, H-L)
( 16B) BDA
Nieuwkoop Wnt8 mRNA VegT mRNA
43
( 16B’)
Wnt8 mRNA
Wnt8 mRNA VegT mRNA
( 16D’, F’, H’, K’, L’)
Wnt8 mRNA (
16C’-E’)
44
16 72
(A, G) B-N-AC AC A Wnt8 mRNABDA G Wnt8 mRNA VegT mRNA
BDA (B-F, B’-F’) A HEBDA HE BDA
(H-L, H’-L’) G HEBDA HE BDA
si, small intestine; mu, muscle; no, notochord; mt, mesothelium; me, mesenchyme 1 mm
45
4.4.
BCNE
Nieuwkoop AC
1)
2)
4.4.1.
B-N-AC AC
BDA Nieuwkoop
BCNE ( 15F’
H’, K’) FGF(Fibroblast growth factor) AC
(Kimelman, 1991;
Faas et al., 2013) Nieuwkoop Nodal
FGF
Nieuwkoop
BCNE ( 15C’
)
BCNE
BCNE
( 15C’-E’) BCNE
BCNE (Kuroda
46
et al., 2004) AC Nodal
BCNE ( 17)
17
B-N-AC ACBCNE ( )
Nieuwkoop ( )BCNE Nieuwkoop
4.4.2.
Wnt8 mRNA BCNE Wnt8
mRNA VegT mRNA Nieuwkoop
Wnt8 mRNA 8~32
(Christian and Moon, 1993)
Wnt8 Wnt8/β-Catenin β-Catenin
BMP Chordin Noggin
(De Robertis and Kuroda, 2004) Wnt8
Wnt8/β-Catenin (Hoppler and
Moon, 1998; Ramel and Lekven, 2004) Wnt8
Wnt8 BCNE
47
Wnt8 mRNA
dnGSK3 β-Catenin Wnt8/β-Catenin
BCNE
48
5 RNA-seq
(Stage 10)
SP-X gain-of-function
RNA CRISPR/Cas
β-Catenin VegT
β-Catenin VegT
Siamois Twin
gRNA PAMmer
AC
BCNE
Nieuwkoop BCNE
iPS
49
(Kuroda et al., 2015)
(Takebe et al.,
2013)
iPS
50
4
2
2
51
1
( :
)
4
52
DeNA
1 4
TA
SFC
!
53
Agius, E., Oelgeschläger, M., Wessely, O., Kemp, C., De Robertis, E.M. (2000).
Endodermal Nodal-related signals and mesoderm induction in Xenopus. Development 127, 1173–1183. [PMID: 10683171]
Ariizumi, T., and Asashima, M. (2001). In vitro induction systems for analyses of
amphibian organogenesis and body patterning. Int. J. Dev. Biol. 45, 273–279. [PMID: 11291857]
Brannon, M., Gomperts, M., Sumoy, L., Moon, R.T., and Kimelman, D. (1997). A
beta-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus. Genes Dev. 11, 2359–2370. [PMID: 9308964]
Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A.,
Somia, N. V, Bogdanove, A.J., and Voytas, D.F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82. [PMID: 21493687]
Chen, B., Gilbert, L. a, Cimini, B. a, Schnitzbauer, J., Zhang, W., Li, G.W., Park, J.,
Blackburn, E.H., Weissman, J.S., Qi, L.S., et al. (2013). Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491. [PMID: 24360272]
Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H., and
Beachy, P. A (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413. [PMID: 8837770]
Chng, S.C., Ho, L., Tian, J., and Reversade, B. (2013). ELABELA: a hormone essential
for heart development signals via the apelin receptor. Dev. Cell 27, 672–680. [PMID: 24316148]
Christian, J.L., and Moon, R.T. (1993). Interactions between Xwnt-8 and Spemann
organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev. 7, 13–28. [PMID: 8422982]
Clements, D., Friday, R.V., and Woodland, H.R. (1999). Mode of action of VegT in
mesoderm and endoderm formation. Development 126, 4903–4911. [PMID: 10518506]
Cordenonsi, M., Dupont, S., Maretto, S., Insinga, A., Imbriano, C., and Piccolo, S.
(2003). Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads. Cell 113, 301–314. [PMID: 12732139]
Cuykendall, T.N., and Houston, D.W. (2009). Vegetally localized Xenopus trim36
regulates cortical rotation and dorsal axis formation. Development 136, 3057–3065. [PMID: 19675128]
De Robertis, E.M., and Kuroda, H. (2004) Dorsal-ventral patterning and neural
induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 295-308. [PMID: 15473842]
54
Faas, L., Warrander, F.C., Maguire, R., Ramsbottom, S. a, Quinn, D., Genever, P., and Isaacs, H. V (2013). Lin28 proteins are required for germ layer specification in Xenopus. Development 140, 976–986. [PMID: 23344711]
Farboud, B., and Meyer, B.J. (2015). Dramatic Enhancement of Genome Editing by
CRISPR/Cas9 Through Improved Guide RNA Design. Genetics 199, 959–971. [PMID: 25695951]
Garneau, J.E., Dupuis, M.È., Villion, M., Romero, D. a, Barrangou, R., Boyaval, P.,
Fremaux, C., Horvath, P., Magadán, A.H., and Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71. [PMID: 21048762]
Gokhale, P.J., and Andrews, P.W. (2008). New insights into the control of stem cell
pluripotency. Cell Stem Cell 2, 4–5. [PMID: 18371412] Harland, R. (2000). Neural induction. Curr. Opin. Genet. Dev. 10, 357–362. [PMID:
10889069] Heasman, J., Kofron, M., and Wylie, C. (2000). Beta-catenin signaling activity
dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222, 124–134. [PMID: 11784070]
Heasman, J., Wessely, O., Langland, R., Craig, E.J., and Kessler, D.S. (2001). Vegetal
localization of maternal mRNAs is disrupted by VegT depletion. Dev. Biol. 240, 377–386. [PMID: 10885751]
Hoff, S., Halbritter, J., Epting, D., Frank, V., Nguyen, T.M., van Reeuwijk, J., Boehlke,
C., Schell, C., Yasunaga, T., Helmstädter, M., et al. (2013). ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3. Nat. Genet. 45, 951–956. [PMID: 23793029]
Hoppler, S., and Moon, R.T. (1998). BMP-2/-4 and Wnt-8 cooperatively pattern the
Xenopus mesoderm. Mech. Dev. 71, 119–129. [PMID: 9507084] Horb, M.E., and Thomsen, G.H. (1999). Tbx5 is essential for heart development.
Development 126, 1739–1751. [PMID: 10079235] Hou, S., Maccarana, M., Min, T.H., Strate, I., and Pera, E.M. (2007). The secreted
serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo. Dev. Cell 13, 226–241. [PMID: 17681134]
Houliston, E., and Elinson, R.P. (1991). Patterns of microtubule polymerization relating
to cortical rotation in Xenopus laevis eggs. Development 112, 107–117. [PMID: 1769322]
Hulstrand, A.M., Schneider, P.N., and Houston, D.W. (2010). The use of antisense
oligonucleotides in Xenopus oocytes. Methods 51, 75–81. [PMID: 20045732] Ishibashi, H., Matsumura, N., Hanafusa, H., Matsumoto, K., De Robertis, E.M., and
Kuroda, H. (2008). Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center is required for brain formation in Xenopus laevis embryos. Mech. Dev. 125, 58–66. [PMID: 18036787]
55
Kenyon, K.L., Moody, S.A., and Jamrich, M. (1999). A novel fork head gene mediates early steps during Xenopus lens formation. Development 126, 5107–5116. [PMID: 10529427]
Kikkawa, M., Takano, K., and Shinagawa, A. (1996). Location and behavior of dorsal
determinants during first cell cycle in Xenopus eggs. Development 122, 3687–3696. [PMID: 9012490]
Kim, Y.G., Cha, J., and Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc
finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160. [PMID: 8577732]
Kinoshita, K., and Asashima, M. (1995). Effect of activin and lithium on isolated
Xenopus animal blastomeres and response alteration at the midblastula transition. Development 121, 1581–1589. [PMID: 7600976]
Kuroda, H., Iwamiya, T., and Morimoto, K. (2015). The Experimental Procedures to
Create Life and Transplantable Heart. SFC J. 15, 262–282. Kuroda, H., Wessely, O., and De Robertis, E.M. (2004). Neural induction in Xenopus:
requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus. PLoS Biol. 2, E92. [PMID: 15138495]
Larraín, J., Oelgeschläger, M., Ketpura, N.I., Reversade, B., Zakin, L., and De Robertis,
E.M. (2001). Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling. Development 128, 4439–4447. [PMID: 11714670]
Macdonald, R., Barth, K. a, Xu, Q., Holder, N., Mikkola, I., and Wilson, S.W. (1995).
Midline signalling is required for Pax gene regulation and patterning of the eyes. Development 121, 3267–3278. [PMID: 7588061]
Mei, W., Jin, Z., Lai, F., Schwend, T., Houston, D.W., King, M. Lou, and Yang, J.
(2013). Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification. Development 140, 2334–2344. [PMID: 23615278]
Michiue, T., and Asashima, M. (2005). Temporal and spatial manipulation of gene
expression in Xenopus embryos by injection of heat shock promoter-containing plasmids. Dev. Dyn. 232, 369–376. [PMID: 15614780]
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S.,
Korinek, V., Roose, J., Destrée, O., and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391–399. [PMID: 8756721]
Nieuwkoop, P.D. (1969). The formation of the mesoderm in Urodelean amphibians. I.
Induction by the endoderm. Roux’ Arch. f. Entw. Mech. 162, 34–373. Nieuwkoop, P.D. (1973). The organization center of the amphibian embryo: its origin,
spatial organization, and morphogenetic action. Adv. Morphog. 10, 1–39. [PMID: 4581327]
56
O’Connell, M.R., Oakes, B.L., Sternberg, S.H., East-Seletsky, A., Kaplan, M., and Doudna, J.A. (2014). Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266. [PMID: 25274302]
Oelgeschläger, M., Larraín, J., Geissert, D., and De Robertis, E.M. (2000). The
evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling. Nature 405, 757–763. [PMID: 10866189]
Okita, K., Hong, H., Takahashi, K., and Yamanaka, S. (2010). Generation of
mouse-induced pluripotent stem cells with plasmid vectors. Nat. Protoc. 5, 418–428. [PMID: 20203661]
Pauli, A., Montague, T.G., Lennox, K.A., Behlke, M.A., and Schier, A.F. (2015).
Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish. PLoS One 10, e0139504. [PMID: 26436892]
Piccolo, S., Sasai, Y., Lu, B., and De Robertis, E.M. (1996). Dorsoventral patterning in
Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589–598. [PMID: 8752213]
Ramel, M.C., and Lekven, A.C. (2004). Repression of the vertebrate organizer by Wnt8
is mediated by Vent and Vox. Development 131, 3991–4000. [PMID: 15269175] Rotman, N., Guex, N., Gouranton, E., and Wahli, W. (2013). PPARβ interprets a
chromatin signature of pluripotency to promote embryonic differentiation at gastrulation. PLoS One. 8, e83300. [PMID: 24367589]
Sho, T., Tsukiyama, T., Sato, T., Kondo, T., Cheng, J., Saku, T., Asaka, M., and
Hatakeyama, S. (2011). TRIM29 negatively regulates p53 via inhibition of Tip60. Biochim. Biophys. Acta 1813, 1245–1253. [PMID: 21463657]
Slack, J.M., Darlington, B.G., Gillespie, L.L., Godsave, S.F., Isaacs, H.V., and Paterno,
G.D. (1989). The role of fibroblast growth factor in early Xenopus development. Development 107 Suppl., 141–148. [PMID: 2636135]
Spemann, H. and Mangold, H. (1924). Induction of embryonic primordia by
implantation of organizers from a different species. Roux’ Arch. Entw. Mech., 100, 599-638. [PMID: 11291841]
Strähle, U., Blader, P., Henrique, D., and Ingham, P.W. (1993). Axial, a zebrafish gene
expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev. 7, 1436–1446. [PMID: 7687227]
Takebe, T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri, T., Zhang, R.R.,
Ueno, Y., Zheng, Y.W., Koike, N., et al. (2013). Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484. [PMID: 23823721]
Tao, Q., Yokota, C., Puck, H., Kofron, M., Birsoy, B., Yan, D., Asashima, M., Wylie,
C.C., Lin, X., and Heasman, J. (2005). Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120, 857–871. [PMID: 15797385]
Tompsett, A.R., Wiseman, S., Higley, E., Giesy, J.P., and Hecker, M. (2013). Effects of
57
exposure to 17α-ethynylestradiol during sexual differentiation on the transcriptome of the African clawed frog (Xenopus laevis). Environ. Sci. Technol. 47, 4822–4828. [PMID: 23550701]
Tran, U., Pickney, L.M., Ozpolat, B.D., and Wessely, O. (2007). Xenopus Bicaudal-C is
required for the differentiation of the amphibian pronephros. Dev. Biol. 307, 152–164. [PMID: 17521625]
Woolf, T.M., Jennings, C.G., Rebagliati, M., and Melton, D.A. (1990). The stability,
toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic Acids Res. 18, 1763–1769. [PMID: 1692405]
Yamanaka, S. (2008). Induction of pluripotent stem cells from mouse fibroblasts by
four transcription factors. Cell Prolif. 41 Suppl. 1, 51–56. [PMID: 18181945] Yuan, Z., Villagra, A., Peng, L., Coppola, D., Glozak, M., Sotomayor, E.M., Chen, J.,
Lane, W.S., and Seto, E. (2010). The ATDC (TRIM29) protein binds p53 and antagonizes p53-mediated functions. Mol. Cell. Biol. 30, 3004–3015. [PMID: 20368352]
Zhang, J., and King, M.L. (1996). Xenopus VegT RNA is localized to the vegetal cortex
during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning. Development 122, 4119–4129. [PMID: 9012531]
Zimmerman, L.B., De Jesús-Escobar, J.M., and Harland, R.M. (1996). The Spemann
organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599–606. [PMID: 8752214]
58
1
https://youtu.be/MFa9TYQOZ1Q
2 SP-X mRNA
https://youtu.be/DdT9KFP1xCE
3
https://youtu.be/fVDPxN7A_ok
https://youtu.be/qiSvVLJpzwo
https://youtu.be/f6gRvLkJuJI
https://youtu.be/ITifNryVnn4
Printed in Japan 印刷・製本 ワキプリントピア
2016年 3 月31日 初版発行
SFC-SWP 2015-010
著者 森本健太
監修 先端生命科学研究会
発行 慶應義塾大学 湘南藤沢学会 〒252-0816 神奈川県藤沢市遠藤5322
TEL:0466-49-3437
アニマルキャップ細胞群を用いた胚全体構造の構築への挑戦
■
本論文は研究会において優秀と認められ、出版されたものです。