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Figure legends

FIGURE 1

Targeting of the chicken PRDM14 locus in PGCs. (a) Structure of CRISPR/Cas9

targeting sites in PRDM14. The upper part of the diagram shows the PRDM14 wild-type

allele. The first 4 exons are shown as boxes, and the 5’-untranslated region is shown by

a white box. The position of the deleted sequence by recombination and the sgRNA

targeting site are indicated by a hatched box and arrowhead, respectively. The bottom

part shows the structure of the knocked-in allele. The left and right homology arms (HA,

dotted squares) and the eGFP coding sequence with a stop codon are indicated. The

primer sets for the original allele were shown in black arrows, and for the left and right

arms in gray and blanked arrows, respectively. (b) Flow cytometric analyses of eGFP

fluorescence after the reporter cassette was knocked into the PRDM14 locus. A flow

cytometric analysis was performed after the two-round sorting of eGFP-positive PGCs.

(c) Targeted gene-trapping using the reporter cassette was detected by genomic PCR in

eGFP knocked-in PGC clones. WT, wild-type genome.

FIGURE 2

Generation of PRDM14 gene-trapped G1 transgenic chickens. (a) Flow cytometric

analysis of the gonadal cells of 5.5-day recipients after grafting eGFP knocked-in PGCs.

20

a and b gates indicate SSEA-1+eGFP− and SSEA-1+eGFP+ cells, respectively. The

control was an ungrafted embryo. (b) Copy numbers of the eGFP sequence in the semen

of germline chimeric chickens. The genomic DNAs of eGFP transgenic chickens (copy

number of 1 (Motono et al., 2010)) were used as a standard. Data are the mean ±

standard error of 5 (c828, c834 and c835) or 2 (c830 and c831) different samples. c833

was from a single sample. (c) Detection of the eGFP reporter cassette in the offspring of

chimeric chicken c835. Genomic DNAs were extracted from the blood of hatched

chicks and subjected to PCR using eGFP-specific primers. GAPDH was amplified as an

internal control. PC (positive control) is the genome of an eGFP transgenic chicken.

Typical results of PCR analyses were shown.

FIGURE 3

Detection of eGFP expression in G1 embryonic PGCs. (a) Flow cytometric analysis of

2.5-day embryonic blood. Blood cells were stained with the anti-SSEA-1 antibody. a

and b gates indicate SSEA-1+eGFP− and SSEA-1+eGFP+ cells, respectively. (b)

Distribution of eGFP-PGCs in the genital ridges of 3.5-day transgenic embryos. The

eGFP fluorescence of dissected trunks as observed from the ventral aspect. Genital

ridge regions were surrounded by dotted lines. (c) Flow cytometric analysis of

eGFP-PGCs in the genital ridges of 3.5-day transgenic embryos. a and b gates indicate

SSEA-1+eGFP− and SSEA-1+eGFP+ cells, respectively.

21

FIGURE 4

PGC-specific expression of eGFP in gonads of transgenic embryos. (a) Flow cytometric

analysis of eGFP-PGCs in the gonads of 6.5-day transgenic embryos. Gonadal cells

were stained with an anti-SSEA-1 antibody. a and b gates indicate SSEA-1+eGFP− and

SSEA-1+eGFP+ cells, respectively. (b) Distribution of eGFP-PGCs in the gonads of

6.5-day

transgenic

embryos

compared

with

non-transgenic

control. (c)

Immunohistochemistry of 6.5-day transgenic gonads. Frozen sections were stained with

the anti-eGFP antibody and anti-SSEA-1 antibody.

FIGURE 5

Expression of eGFP and PRDM14 in PRDM14 gene-trapped embryos. RNAs were

purified from the blastodermal cells of PRDM14 gene-trapped embryos and subjected to

qRT-PCR. The expression levels of eGFP and PRDM14 are shown as relative

expression levels against GAPDH. Data are the mean ± standard error (N=19 (+/+), 23

(+/eGFP), 7 (eGFP/eGFP)). * indicates significant differences by the Student’s t-test

(p<0.01).

22

(a) 500 bp cPRDM14 2 1 WT Left-HA (797 bp) Right-HA (779 bp) eGFP Gene-trapped HindⅢ 1 2 3 4 stop WT Gene-trapped Auto (b) 4 3 0.2% 93.38% 0.02% eGFP (c) PGC clone number WT #1

original allele #2

#3

#4 (980 bp) Gene-trapped left arm (1435 bp) Gene-trapped right arm (1366 bp) Fig. 1

PE (SSEA-1) (a) #1 a b #2 a b a : 0.24%

b : 1.44% #3 a a : 0.55%

b : 0.61% control b a b a : 0.74%

b : 3.26% a : 2.35%

b : 0.01% eGFP (b) 1.4

Copy number 1.2

0.8

0.6

0.4

0.2

c828

(c) c830

c831

c833

c834

c835

hatched chicks 1 2 3 4 5 6 7 8 PC water eGFP (187 bp) GAPDH (182 bp) Fig. 2

(a) 2.5-day blood WT B b A a B b PE (SSEA-1) A a TG

eGFP (b) Genital ridge eGFP WT 3.5-day embryo TG

(c) WT a A B b TG

a B b PE (SSEA-1) A 3.5-day genital ridge eGFP Fig. 3

(a) 6.5-day gonad WT TG a PE (SSEA-1) a b b eGFP (b) WT TG eGFP SSEA-1 DAPI eGFP/SSEA-1 (c) Fig. 4

eGFP

PRDM14

*p=3.4E-03 *p=4.8E-10 *p=4.1E-09 *p=5.4E-04 0.0012

Relative expression (/GAPDH)

Relative expression (/GAPDH)

0.08

0.07

0.06

0.05

0.04

0.03

0.02

0.01

*p=1.5E-03 *p=4.7E-03 0.001

0.0008

0.0006

0.0004

0.0002

+/+

+/+

+/+/eGFP

-/eGFP/eGFP

+/+

+/+

+/+/eGFP

-/eGFP/eGFP

Fig. 5

Table 1: Genotyping of PRDM14 in G2 embryos

PRDM14+/+ embryo (%)

PRDM14+/eGFP embryo (%) PRDM14eGFP/eGFP embryo (%)

blastoderm

27 (33.3%)

38 (46.9%)

16 (19.8%)

2.5 days

9 (29.0%)

22 (71.0%)

3.5 days

12 (32.4%)

25 (67.6%)

Table 1

Supplementary Table 1: Primer List

Primers for HA cloning and sgRNA

PRDM14 genome

Dir

CATGGATCCAAATCAGGGCTTTGGAGGATGGC

Rev CATCTCGAGCTCCCACATCAGCGAGTTGTCG

PRDM14 sgRNA

Dir

CACCGCGTCCGTGGCGTCCGTGGC

Rev AAACGCCACGGACGCCACGGACGC

(Restriction enzymes recognition sites were underlined)

Primers for qRT-PCR and genomic DNA analysis

GAPDH

Dir

GGGCACGCCATCACTATC

Rev GTGAAGACACCAGTGGACTCC

eGFP

Dir

CGGCAACTACAAGACCCGC

Rev GAAGTTCACCTTGATGCCGTTC

PRDM14*

Dir

TACAGCGACTCCTCGCCTTT

Rev GTCCGTGGCGTCCGTG

*, This primer set amplifies only endogenous PRDM14.

Primers for confirming genome editing

CACAGCTCAGGCTCAGGGTTTC

original allele

Dir

Rev CGAGCGCGTCCGTGGCGT

left arm**

Rev GAAGTTCACCTTGATGCCGTTC

CGGCAACTACAAGACCCGC

right arm

Dir

Rev TCAGCTGCCCGTACTCGAAGAC

**, used in combination with direct primer for original allele.

Table. S1

...