This is the pre-peer reviewed version of the following article: Genesis 58: e23388 (2020), which has
been published in final form at doi.org/10.1002/dvg.23388. This article may be used for
non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived
Versions.
Primordial germ cell-specific expression of eGFP in transgenic chickens

Yota Hagihara1*, Yuya Okuzaki1,2*, Kazuma Matsubayashi1, Hidenori Kaneoka1**,
Takayuki Suzuki2,3, Shinji Iijima1, and Ken-ichi Nishijima1,2,3

1

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya

University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan
2

Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences,

Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan
3

Laboratory of Avian Bioscience, Graduate School of Bioagricultural Sciences, Nagoya

University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan*These authors contributed equally**Corresponding author: Department of Biomolecular Engineering, Graduate School ofEngineering, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan
Fax: +81 52 789 3221
E-mail: kaneoka@chembio.nagoya-u.ac.jp

1Funding information
This work was partly supported by Grants-in-Aid for Scientific Research (KAKENHI)(Grant Numbers 16H01253, 16H04574, and 17K14867) and the Kieikai Research
Foundation.

2

Abstract
PR domain zinc finger protein 14 (PRDM14) plays an essential role in thedevelopment of primordial germ cells (PGCs) in mice. However, its functions in avian
species remain unclear. In the present study, we used CRISPR/Cas9 to edit the
PRDM14 locus in chickens in order to demonstrate its importance in development. The
eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs toknockout PRDM14 and label PGCs. Chimeric chickens were established by a direct
injection of eGFP knocked-in (gene-trapped) PGCs into the blood vessels ofHamburger-Hamilton stages (HH-stages) 13-16 chicken embryos. Gene-trappedchickens were established by crossing a chimeric chicken with a wild-type hen withvery high efficiency. Heterozygous gene-trapped chickens grew normally and
SSEA-1-positive cells expressed eGFP during HH-stages 13-30. These results indicatedthe specific expression of eGFP within circulating PGCs and gonadal PGCs. At theblastodermal stage, the ratio of homozygous gene-trapped embryos obtained by crossing
heterozygous gene-trapped roosters and hens was almost normal; however, all embryos
died soon afterward, suggesting the important roles of PRDM14 in chicken early
development.

Keywords
Chicken; CRISPR/Cas9; Primordial germ cells; PRDΜ14; eGFP

3

1. Introduction
Transgenic chickens have been constructed by many groups, including ours, using
retro- or lentiviral vectors (Kamihira et al., 2005; McGrew et al., 2004; Motono et al.,2010; Zhu et al., 2005). However, in most cases, the germinal transmission oftransgenes has been very low, which makes the establishment of transgenic chickens alabor- and time-consuming process. Rapid advances in genome editing technology,
particularly that using transcription activator-like effector nuclease (TALEN) andclustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associatedprotein 9 (Cas9), have provided a simple and convenient process for the establishmentof transgenic chickens using cultured primordial germ cells (PGCs). DDX4- andovomucoid-knockout chickens have already been established by these technologies(Oishi et al., 2016; Taylar et al., 2017). Thus, PGCs are very important for chicken
genetic engineering; however, their growth and differentiation mechanisms have not yet
been elucidated in detail, thereby limiting the efficiency of manipulations.
PR domain zinc finger protein 14 (PRDM14) is a key regulator of germinal fate andis expressed in PGCs. In mice, Prdm14 has been shown to function together with
Blimp1 and AP2γ, and repress mesodermal genes in PGCs. In addition, theco-expression of these factors in P19 embryonal carcinoma cells has been suggested toinduce PGC-like cells in an in vitro model (Kurimoto et al., 2008; Magnúsdóttir et al.,4

2013; Yamaji et al., 2008). However, limited information is currently available on thefunction of chicken PRDM14. We previously reported that the higher expression of
PRDM14 was specific to PGCs, except for pluripotent cells at the blastodermal stage
and neural plate cells at Hamburger Hamilton stage (HH-stage) 4, as well as to the adulttestis. Furthermore, PRDM14 was essential for the proper development of PGCs in vivo
(Okuzaki et al., 2019). However, the role of chicken PRDM14 in early embryonicdevelopment remains unknown. We herein report the establishment of an eGFP
knocked-in (gene-trapped) chicken targeting the PRDM14 locus using the
CRISPR/Cas9 system. The specific expression of eGFP in PGCs was observed inheterologous gene-trapped embryos. Furthermore, an analysis of its homozygous
descendants demonstrated that PRDM14 is indispensable for early embryonicdevelopment.

2. Results and discussion

To clarify the function of PRDM14 using gene-trapped chickens, we designed agenome-editing strategy. CRISPR/Cas9-mediated homologous recombination was used
to target the PRDM14 locus; the eGFP gene containing the stop codon was inserted
immediately downstream of the initiation codon of PRDM14 based on the sequence wepreviously reported (Okuzaki et al., 2019). The targeting vector had a 797-bp sequence5

containing a 5’-untranslated region plus a coding sequence of 12 nucleotides as the
5’-homology arm, and a 779-bp sequence that contained a part of exon 1 and the entire
sequence from intron 1 to exon 3 as the 3’-homology arm. The eGFP gene flanked byadditional HindIII linker sequences was inserted between these homologous arms (Fig.
1a). Three single-guide RNAs (sgRNAs) were designed based on the 5’-end region of
the PRDM14 coding sequence using CRISPR-direct (https://crispr.dbcls.jp) and one
sgRNA sequence was selected based on their recombination activity checked by asingle-strand annealing assay. No candidate sequence for off-target sites was predicted
by CRISPR-direct for sgRNA.
Cas9 and sgRNA expression and targeting vectors were simultaneously introduced
into cultured chicken PGCs, established from a male embryo, by electroporation. Fivedays post-transfection, eGFP-positive cells were collected using a cell sorter. The
percentage of eGFP-positive cells was 1.25%. This percentage increased toapproximately 77% by the first sorting and the copy number of eGFP in bulk cells wasapproximately 0.7. After two-cycle purification by a cell sorter, the fraction of
eGFP-positive cells increased to approximately 93% (Fig. 1b). We also isolated several
clones and their copy numbers of eGFP were almost 1 (data not shown), suggesting thata single copy of the eGFP gene was introduced into the diploid genome. This was
confirmed by PCR using a specific primer. Since homologous recombination resulted ina 33-bp deletion in exon 1, a primer was designed in this deletion sequence for the6specific amplification of the original allele (Fig. 1a). Amplification from the original
allele was observed in all samples, while the left and right arms of the transgene were
amplified from the PGC clones, but not from wild-type PGC (Fig. 1c). Thus, all of the
PGC clones obtained were heterozygous gene-trapped cells. The lack of homozygous
gene-trapped clones was consistent with our previous findings showing that PRDM14
was required for the proliferation of PGCs in vivo (Okuzaki et al., 2019).
eGFP-positive PGCs purified by flow cytometry (not cloned) were transplanted intodeveloping 2.5-day embryos as a preliminary trial. After a 3.5-day in vitro culture ofgrafted embryos, the percentages of SSEA-1+eGFP+ cells in the gonad were similar or
higher than SSEA-1+eGFP cells (Fig. 2a). The replacement rates in grafted embryo #1,−#2, and #3 were 85.7, 52.6, and 81.5%, respectively, suggesting that manipulated PGCs
settled in the gonads with high efficiency. We obtained 6 male germline chimericchickens from the in vitro culture of manipulated embryos. After sexual maturation, thecopy number of the transgene in the sperm was analyzed by qPCR. The copy number
varied by individuals and one chicken (c835) showed the highest copy number ofapproximately 0.9 (Fig. 2b). Following the artificial insemination of a wild-type henwith c835 semen, 18 transgenic chickens out of 84 descendants were obtained (Fig. 2c).
Their copy numbers of eGFP were almost 1 (data not shown) and sequence analyses ofgenomic DNA from the blood cells of a G1 transgenic chicken showed that the eGFP
gene was inserted into the expected site.

7In order to confirm the expression of eGFP, the blood of 2.5-day embryos from G1chickens was analyzed using a flow cytometer. SSEA-1+eGFP+ cells were detected intransgenic chickens, but not in wild-type blood cells (Fig. 3a), suggesting thatcirculating PGCs (cPGCs) expressed eGFP. In the genital ridges of 3.5-day embryosfrom a G1 chicken, we detected eGFP-positive cells using a fluorescence microscope(Fig. 3b). Flow cytometric analyses showed that eGFP-positive cells were also positive
for SSEA-1 in the genital ridge of a transgenic chicken; however, SSEA-1+eGFP+ cells
were not detected in those of wild-type chickens (Fig. 3c). These results suggested that
PGCs in the genital ridge expressed eGFP. In addition, SSEA-1+eGFP+ cells weredetected in the gonadal cells of a 6.5-day transgenic chicken by flow cytometricanalyses, but not in wild-type embryos (Fig. 4a). Fluorescent images offrozen-sectioned gonads showed that eGFP fluorescence was mainly observed in thecortex of gonads (Fig. 4b), which corresponds to the site of accumulation of PGCs inthis stage (Ayers et al., 2013). Immunostaining showed that the majority of
SSEA-1-positive cells in the gonads expressed eGFP, suggesting that eGFP was
specifically expressed in the PGCs of the gonads (Fig. 4c), but not in SSEA-1-negative
cells, which contain both somatic and differentiated germ cells at this stage.
Collectively, these results show that eGFP is expressed in PGCs in embryonic bloodand the gonads of G1 transgenic chicken, reflecting the expression pattern ofendogenous PRDM14. Therefore, PGCs can be detected by eGFP fluorescence.

8In order to investigate the function of PRDM14 in embryonic development, G2
descendants were obtained by mating between G1 hens and roosters. The appearance of
wild-type (PRDM14+/+) as well as PRDM14 heterozygous (PRDM14+/eGFP) andhomozygous gene-trapped (PRDM14eGFP/eGFP) chickens was analyzed at various
developmental stages. PRDM14eGFP/eGFP embryos were detected at the blastodermal
stage at a slightly lower frequency than Mendelian predictions (Table 1). A qRT-PCR
analysis showed that the expression level of eGFP in PRDM14+/eGFP embryos was 50%
that in PRDM14eGFP/eGFP. PRDM14 in PRDM14+/eGFP embryos was 50% that in thewild-type, and PRDM14 was not detected in PRDM14eGFP/eGFP embryos, as expected(Fig. 5). However, after a 2.5-day incubation, PRDM14eGFP/eGFP embryos did not survive(Table 1), suggesting the important role of PRDM14 in the early development ofchickens. Furthermore, we did not observe any PRDM14eGFP/eGFP embryos after
HH-stage 3 (data not shown). This was unexpected because mice lacking Prdm14 werepreviously reported to be sterile, but viable (Yamaji et al., 2008), and the knockout of
PRDM14 in Zebrafish did not cause early embryonic lethality, but resulted in motor
neuron maturational arrest (Liu et al., 2012). We previously showed that PRDM14 was
expressed in cells other than PGCs, such as in the blastoderm (Okuzaki et al., 2019),
and the loss of PRDM14 expression in these cells may have caused early embryoniclethality in PRDM14eGFP/eGFP chickens.
We previously demonstrated that PRDM14 was essential for the proliferation of

9

PGCs after the blastoderm because the number of PGCs was decreased by its in vivo
knockdown during these stages (Okuzaki et al., 2019). We failed to confirm that
PRDM14 knockout decreased PGCs because of the embryonic lethality of
PRDM14eGFP/eGFP. Further detailed studies, such as those using an inducible and/or
cell-specific knockout system for PRDM14 expression, are needed to clarify the stagesof PGCs and/or the cells at specific stages that critically required PRDM14 expression
for proper development.

3. Methods

3.1. Chickens and eggs
Fertilized eggs (White Leghorn) were purchased from Nisseiken or Japan Layer.
All animal experiments were performed according to the Ethical Guidelines for Animal
Experimentation of Nagoya University.

3.2. PGC culture
Blood containing cPGCs was collected from embryos at HH-stages 13-16 and cells
from a single embryo were cultured in PGC medium containing KO-DMEM (Thermo
Fisher Scientific) supplemented with B-27 supplement (Thermo Fisher Scientific, 1×),L-glutamine (Wako, 2 mM), non-essential amino acids (Wako, 1×), 0.1 mM

10

2-mercaptoethanol (Wako), nucleotides (Thermo Fisher Scientific, 1×), 100 U/ml
penicillin G (Wako), 100 µg/ml streptomycin (Wako), 22 U/ml sodium heparin (Wako),
1.2 mM sodium pyruvate (Thermo Fisher Scientific), chicken serum (0.2%), activin A(Shenandoah Biotechnology, 25 ng/ml), BMP4 (Peptide Institute, 25 ng/ml), and FGF2
(PeproTech, 4 ng/ml). These PGC preparations were derived from a male embryo andalmost all cells were SSEA-1- and chicken Vasa homolog (CVH)-positive (Okuzaki etal., 2019).

3.3. Plasmid construction
The plasmid expressing Cas9 and sgRNA was generated by inserting annealed andphosphorylated oligonucleotides corresponding to the targeting sequence (Table S1)
into the BbsI site of pX330 (Addgene). In the construction of the targeting vector, thegenomic DNA sequence of PRDM14 was amplified by PCR from the chicken genome
as a template with the primers (Table S1). The amplified fragment was cloned into
pGL3-basic (Promega). The eGFP coding sequence was then inserted immediately after
the start codon of PRDM14 in-frame to disrupt the PRDM14 protein.

3.4. Transfection and single-cell cloning of PGCs
Cas9/sgRNA expression and targeting vectors were introduced into 1.5 × 105 cellsof cultured PGCs by electroporation using the electroporator NEPA21 (NEPAGENE).

11

PGCs were then transferred to PGC medium (Whyte et al., 2015) containing 10 ng/ml
Y27632 (Wako) in a 24-well plate. After a one-week culture, eGFP-positive cells wereisolated using the flow cytometer FACSJazz (BD Bioscience) and then transferred to a
96-well plate for cloning.

3.5. Genomic DNA analysis
Genomic DNA was purified from PGCs and chicken semen using Mag
Extractor-Genome- (TOYOBO). To confirm the presence of the knock-in fragment,
targeting sites were amplified with several sets of primers (Table S1), and the copy
number of eGFP was measured by LightCycler 96 (Roche Diagnostics) using the
genomic DNA of the eGFP transgenic chicken as a control (Motono et al., 2010).

3.6. Analysis of grafted PGCs in gonads of 5.5-day recipientsTo assess the proportion of donor-derived PGCs that had migrated and settled in the
gonads of the recipients, 5,000 eGFP-positive PGCs were injected into the blood vesselsof HH-stages 13-16 recipient embryos in bulk as described previously (Motono et al.,2010). After an incubation for 3.5 days, recipient embryos were washed withphosphate-buffered saline (PBS) and the gonads were dissected. Cells from the gonads
were incubated with an anti-SSEA-1 antibody (Santa Cruz Biotechnology) at 4˚C for 1
h. After washing with PBS, cells were incubated with phycoerythrin (PE)-labeled goat12

anti-mouse IgM (Santa Cruz Biotechnology) at 4˚C for 30 min. The percentages of
SSEA-1+eGFP+ cells were measured using a flow cytometer.

3.7. Production of germline chimeric chickens
To produce germline chimeras, approximately 5,000 eGFP-positive PGCs were
injected into male embryos as described above. Embryos were cultured and allowed tohatch as previously reported (Motono et al., 2010).

3.8. Progeny test
Germline chimeric chickens were mated with wild-type hens after sexual maturation.
Genomic DNA from the blood of offspring was extracted using Easy DNA Extraction
kit Version 2 (KANEKA). The primers for PCR are shown in Table S1.
Heterozygous gene-trapped chicken line (PRDM14+/eGFP) will be deposited at Avian
Bioscience Research Center and made available to the research community uponrequest.

3.9. Observation of eGFP expression in PGCs of heterozygous G1 transgenic chickens
by a flow cytometric analysis and fluorescent microscopy
Blood containing cPGCs was collected from the 2.5-day embryos of G1 transgenic
chickens, and cells were indirectly stained with an anti-SSEA-1 antibody. The

13

percentages of SSEA-1+eGFP+ cells were measured using a flow cytometer. To
examine the localization of eGFP-positive PGCs, the genital ridges of 3.5-day embryos
were observed from the ventral aspect by a fluorescence microscope (Leica
Microsystems).

3.10. Immunohistochemistry
The gonadsof

6.5-day embryos

were dissected and fixed with 4%paraformaldehyde (PFA) at 4˚C for 2 h. The gonads were treated stepwise with 10, 20,
and 30% sucrose in PBS for 1 h at each concentration on ice and then embedded in acompound for frozen sections (Leica Microsystems). Samples were sectioned by acryostat (Leica Microsystems) for immunohistochemistry. Cells in frozen sections werepermeabilized by a treatment with 0.2% Triton X-100 (Wako) at room temperature for
20 min, and then blocked with 3% bovine serum albumin (BSA) in PBS for 30 min.
Samples were incubated at 4°C overnight with primary antibodies (anti-GFP (Sigma) or
anti-SSEA-1) followed by secondary antibodies (anti-mouse IgG-Alexa Fluor 488(Thermo Fisher Scientific) or anti-mouse IgM-PE) at room temperature for 2 h. After an
incubation with 100 ng/ml of DAPI in PBS for 3 min, fluorescent images were takenwith an upright microscope (Leica Microsystems).

3.11. qRT-PCR

14

Total RNA was extracted from blastodermal cells using the ReliaPrep RNA Cell
Miniprep System (Promega), and the ReverTra Ace qPCR RT Master Mix with gDNA
Remover (TOYOBO) were used for cDNA synthesis. Real-time PCR was performed
using LightCycler 96 and Thunderbird qPCR Mix (TOYOBO). LightCycler
amplification involved pre-denaturation at 95°C for 60 s, followed by 40 cycles of 95°C
for 3 s, 60°C for 10 s, and 72°C for 30 s. The primers used are listed in Table S1.

Conflicts of Interest
The authors declare no conflicts of interest associated with this manuscript

Acknowledgments
We thank Dr. Y. Yoshida (Innovative Research Center for Preventive Medical
Engineering, Nagoya University) for the use of FACSJazz.

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18

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

diagramshows

the PRDM14 wild-type allele. The first 4 exons are shown as boxes, and the5’-untranslated region is shown by a white box. The position of the deleted sequence byrecombination and the sgRNA targeting site are indicated by a hatched box andarrowhead, 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 sequencewith a stop codon are indicated. The primer sets for the original allele were shown inblack 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 thetwo-round sorting of eGFP-positive PGCs. (c) Targeted gene-trapping using thereporter 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 cytometric19

analysis of the gonadal cells of 5.5-day recipients after grafting eGFP knocked-in PGCs.
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 semenof germline chimeric chickens. The genomic DNAs of eGFP transgenic chickens (copynumber 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 ofchimeric chicken c835. Genomic DNAs were extracted from the blood of hatchedchicks and subjected to PCR using eGFP-specific primers. GAPDH was amplified as aninternal 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. aand 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
dissected trunks of embryos were 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

20−

and SSEA-1+eGFP+ cells, respectively.

FIGURE 4
PGC-specific expression of eGFP in gonads of transgenic embryos. (a) Flow cytometricanalysis 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-daytransgenic

embryos

compared

withanon-transgenic

control.

(c)

Immunohistochemistry of 6.5-day transgenic gonads. Frozen sections were stained withthe 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 toqRT-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).

21

(a)500 bpcPRDM14

WT

1

2

Left-HA (797 bp)

Right-HA (779 bp)

eGFP

Gene-trapped

1

HindⅢ

(b)

4

3

2

3

4

stop

Gene-trapped

Auto

WT

0.2%

93.38%

0.02%

eGFP

(c)

PGC clone number
WT #1

#2

#3

#4

original allele(980 bp)

Gene-trapped left arm(1435 bp)

Gene-trapped right arm(1366 bp)
Fig. 1

PE (SSEA-1)

(a)

a

#1

b

a

#2

b

a : 0.24%
b : 1.44%#3aa : 0.55%
b : 0.61%
controlbaba : 0.74%
b : 3.26%

a : 2.35%
b : 0.01%
eGFP

(b)

1.4

Copy number

1.2
1
0.8
0.6
0.4
0.2
0
c828

c830

(c)

c831

c833

c834

c835

hatched chicks
1

2

3

4

5

6

7

8

9

10

11 PC watereGFP(187 bp)

GAPDH(182 bp)

Fig. 2(a)2.5-day blood

WT
bB

Aa

B
b

PE (SSEA-1)

Aa

TG

eGFP

(b)

Genital ridge

eGFP

WT

3.5-day embryo
TG

(c)
WT
a

Bb

TG
A

a

bB

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-1DAPI

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
0

+/+
+/+

+/eGFP+/-eGFP/eGFP-/-*p=1.5E-03 *p=4.7E-03

0.001
0.00080.00060.00040.00020+/++/++/+/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%)0

3.5 days

12 (32.4%)

25 (67.6%)0

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 GTGAAGACACCAGTGGACTCCeGFP
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 GAAGTTCACCTTGATGCCGTTCCGGCAACTACAAGACCCGC
right arm
Dir
Rev TCAGCTGCCCGTACTCGAAGAC**, used in combination with direct primer for original allele.

Table. S1