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Cell Stem Cell, Volume 8
Supplemental Information
Defects in Trophoblast Cell Lineage Account for
the Impaired In Vivo Development of Cloned Embryos
Generated by Somatic Nuclear Transfer
Jiangwei Lin, Linyu Shi, Man Zhang, Hui Yang, Yiren Qin, Jun Zhang, Daoqing Gong, Xuan
Zhang, Dangsheng Li, and Jinsong Li
Inventory of Supplemental Information
1. Supplemental Figures and Legends
Figure S1, related to Figure 1 and Table 1
Figure S2, related to Figure 1 and Table 1
2. Table S1, related to Figure S2
3. Supplemental Experimental Procedures
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Figure S1. Distribution of NT-Derived Cells and Tetraploid Cells at
Blastocyst Stage and Later Stages of NTICM/FD4N (1:2) Embryos,
related to Figure 1 and Table 1.
(A) Wholemount confocal image of NTICM/FD4N blastocyst. From left
to right: green fluorescence (marker of cloned-derived cells), red
fluorescence (immunostaining with antibody against Oct4), blue
fluorescence (nuclear dye Hoechst 33342) and overlay.
(B) Total cell number of ICM (Oct4 positive cells) and cell number of
cloned-derived ICM (Oct4-GFP double-positive cells) (n=10).
(C) Wholemount confocal image of NTICM/FD4N blastocyst. From left
to right: green fluorescence (marker of cloned derivatives), red
fluorescence (immunostaining with antibody against Cdx2), blue
fluorescence (nuclear dye Hoechst 33342) and overlay.
(D) Total cell number of trophectoderm (Cdx2 positive cells) and cell
number of trophoblast cells derived from cloned ICM (Cdx2-GFP
double-positive cells) (n=10).
(E) Fetus and placenta collected on Day 12.5 of gestation. The fetus is
strongly green fluorescent. Arrow indicates non-GFP-expressing
extra-embryonic tissues, which are mostly from tetraploid embryos.
(F) Section through placenta of 12.5 dpc. Green-fluorescent cells highly
contributed to labyrinth and chorionic plate of placenta. Scale bar,
500µm
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(G) Wholemount immunostaining image of fetus collected on Day 12.5 of
gestation, fluorescence for GFP and Hoechst33342. Arrows indicate
tetraploid derivatives (GFP-negative cells). Box is magnified in H.
Scale bar, 500µm.
(H) GFP-negative cells. Scale bar, 50µm.
(I) Mouse and placenta collected on Day 19.5 of gestation. The mouse is
strongly green fluorescent. Arrow indicates the placenta, in which,
only a small region displayed green fluorescent (arrowhead).
(J) Section through placenta of 19.5 dpc. Green-fluorescent cells were
observed in labyrinthine layer and chorionic plate of placenta. Scale
bar, 500µm.
(K) The presence of non-GFP-expressing cells in the gut of one clonal
mouse. Box is magnified in L. Scale bar, 100µm.
(L) GFP-negative cells. Scale bar, 10µm.
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Figure S2. Characterizations of the Cloned or Clonal mice, related to
Figure 1 and Table 1.
(A) Adult clonal mice derived from aggregated embryos between one
cloned ICM (wild-type B6D2F1 background) and two tetraploid
embryos (ICR background) exhibit black coat. Arrow indicates a
small batch of white hairs displayed in one of 29 survived clonal mice
from NTICM/FD4N (1:2), reflecting the contamination of tetraploid
cells (ICR) in clonal mice at very low frequency.
(B) Clonal mice derived from aggregated embryos between one cloned
ICM (GFP-transgenic B6D2F1 background) and two tetraploid
embryos (wild-type B6D2F2 background) show strongly green
fluorescent. Arrow indicates non-GFP-expressing placenta, which are
mostly derived from tetraploid embryos.
(C) SSLP analyses were performed using genomic DNA isolated from
cloned mice (from one-step NT) and clonal mice (from
NTICM/FD4N). Genomic DNA from B6D2F1, ICR, C57BL/6 and
DBA mice served as control. C-1 and C-2 denote the cloned mice
from one-step NT. T-1 and T-2 denote the clonal mice from
NTICM/FD4N.
(D) A cloned mouse placenta stained by hematoxylin-and-eosin (HE)
reveals expansion of spongiotrophoblast layer. Yellow broken lines
are the boundary between the spongiotrophoblast layer and labyrinth
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layer.
(E) A placenta derived from NTICM/FD4N aggregated embryo stained
by HE shows a normal-looking histology.
(F) A FD control placenta stained by HE.
(G) Compared with FD placentae, transcription levels of three imprinted
genes (Igf2r, Peg1 and Grb10/Meg1) and four nonimprinted genes
(Vegfr2, Igfbp2, Esx1 and Cpe) are significantly reduced in cloned
placentae (n=8), but greatly increased in placentae (n=8) from
NTICM/FD4N. Bottom left, two imprinted genes (Igf2 and Dlk1) are
normally expressed in placentae derived from different groups.
Bottom right, transcription levels for two imprinted genes (Meg3/Gtl2
and Dcn) are significantly lower in cloned placentae compared with
FD controls, and not significantly improved in placentae from
NTICM/FD4N.
(H) Transcription levels for imprinted genes (H19, Grb10/Meg1, Peg1,
Igf2, Igf2r, Meg3/Gtl2, Dlk1 and Dcn) and kidney-specific gene
(Cdh16) in cloned (n=4) and clonal (n=4, from NTICM/FD4N)
kidney. Three imprinted genes (Igf2, Igf2r, and Dcn) are normally and
four genes (including three imprinted genes: H19, Grb10/Meg1 and
D1K1and the kidney-specific gene: Cdh16) are aberrantly expressed
in both cloned and clonal kidney. The transcript levels for the other
two imprinted genes (Peg1 and Meg3/Gtl2) are significantly
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increased in clonal kidney.
(I) Transcription levels for imprinted genes (H19, Grb10/Meg1, Peg1,
Igf2, Igf2r, Meg3/Gtl2, Dlk1 and Dcn) and heart-specific gene (Myh6)
in cloned (n=4) and clonal (n=4, from NTICM/FD4N) heart. Three
imprinted genes (Igf2, Meg3/Gtl2 and Dcn) are normally and one
imprinted gene (Peg1) is aberrantly expressed in both cloned and
clonal heart. Two imprinted genes (H19 and Grb10) are aberrantly
expressed in clonal heart and three genes (including two imprinted
genes: Igf2r and Dlk1 and heart-specific gene: Myh6) are abnormally
expressed in cloned heart.
(J) Transcription levels for imprinted genes (H19, Grb10/Meg1, Peg1,
Igf2, Igf2r, Meg3/Gtl2, Dlk1 and Dcn) and liver-specific gene (Alb) in
cloned and clonal liver. Six imprinted genes (H19, Grb10/Meg1,
Peg1, Igf2, Igf2r and Meg3/Gtl2) and the liver-specific gene (Alb) are
normally and one imprinted gene (Dlk1) is aberrantly expressed in
both cloned and clonal liver. The other one imprinted gene (Dcn) is
abnormally expressed in clonal liver.
Scale bar, 200 µm (D-F); *: 0.01<p<0.05; **: 0.01<p<0.001; ***:
p<0.001.
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Table S1. Oligonucleotide Primers Used for Quantitative RT-PCR,
related to Figure S2.
Target genes
Primer sequence (Forward) Primer sequence (Reverse)
Igf2r TAGTTGCAGCTCTTTGCACG ACAGCTCAAACCTGAAGCG Peg1 GGCTCCTCTATGATGGCCG AAGCCTTTCTGAACAGCCAGC Vegfr2 GGCAGGTCTGAGGGTTCTCT
G CAAAGCTAAAATACTGAGGACTTGGG
Igfbp2 CCTCAAGTCAGGCATGAAGGA
GCAGGGAGTAGAGATGTTCCA
Esx1 TTGCTAGAGTGGAGCTTGCC GCCTAAATGGTGGAGGCATTC Cpe TGAGAAAGAAGGCGGTCCT
A TTTGGAAAATTGCGTCATCA
Igf2 CTAAGACTTGGATCCCAGAACC
GTTCTTCTCCTTGGGTTCTTTC
Dlk1 ACTTGCGTGGACCTGGAGAA
CTGTTGGTTGCGGCTACGAT
Meg3/Gtl2 TTGCACATTTCCTGTGGGAC AAGCACCATGAGCCACTAGG Dcn CATACTCAAATAAGGCTTCA
CCAA AAAGTTGTCTGTAGTTGTGAACTGA
β-Actin AAGTGTGACGTTGACATCCG
GATCCACATCTGCTGGAAGG
H19 CATGTCTGGGCCTTTGAA TTGGCTCCAGGATGATGT Grb10/Meg1
TCCAAGTGGAGAGTACCATGC
TACGGATCTGCTCATCTTCG
Cdh16 CTCCAGCCTCTCAACGATTCC
GGTGCGTGAACACAAGAATGA
Myh6 GCCCAGTACCTCCGAAAGTC GCCTTAACATACTCCTCCTTGTC
Alb AAGACCCCAGTGAGTGAGCATG
GCTTGTGCTTCACCAGCTCAGC
Gapdh CACTCTTCCACCTTCGATGC CTCTTGCTCAGTGTCCTTGC
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Nuclear Transfer
Nuclear transfer was performed as described (Wakayama et al., 1998)
with modifications (Li et al., 2004; Kishigami et al., 2006). Briefly,
metaphase II-arrested oocytes were collected from superovulated B6D2F1
females (8-10 wks) and cumulus cells were removed using hyaluronidase.
The oocytes were enucleated in a droplet of HEPES-CZB medium
containing 5 µg/ml cytochalasin B (CB) using a blunt Piezo-driven pipette.
After enucleation, the spindle-free oocytes were washed extensively and
maintained in CZB medium up to 2 h before nucleus injection. The CCs
from wild-type or GFP transgenic mice (B6D2F1) were aspirated in and
out of the injection pipette to remove the cytoplasmic material and then
injected into enucleated oocytes. The reconstructed oocytes were cultured
in CZB medium for 1 h and then activated for 5-6 h in activation medium
containing 10 mM Sr2+, 5 ng/ml trichostatin A (TSA) and 5 µg/ml CB.
Following activation, all of the reconstructed embryos were cultured in
KSOM medium supplemented with 5 ng/ml TSA for another 3-4 hours
and maintained in KSOM medium with amino acids at 37 ºC under 5%
CO2 in air.
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Derivation of Tetraploid Embryos
For providing fertilization-derived (FD) tetraploid embryos, ICR or
B6D2F1 females were superovulated and mated with ICR or B6D2F1
males, respectively. Two-cell stage embryos were obtained 44 h after
HCG injection. To generate tetraploid embryos, we electrofused two-cell
embryos to produce one-cell tetraploid embryos. Two-cell embryos were
aligned using an alternating current in 0.3 M mannitol solution, and a
single direct current pulse of 1 000 V/cm was applied for 20 µs. After
electrofusion, the embryos were returned to KSOM medium with amino
acids, and the fused embryos were cultured for one day to reach the
four-cell stage.
Isolating the ICM of Cloned or FD Blastocysts
ICM was isolated by immunosurgery as described (Solter and Knowles,
1975). The zona pellucida of cloned or FD blastocysts was removed using
acid Tyrode solution. Blastocysts were incubated with rabbit anti-mouse
serum (Sigma, diluted 1:1 with KSOM) for 30 minutes. After washed
thoroughly, all blastocysts were exposed to guinea pig serum (diluted 1:1
with KSOM) for 30 minutes. The outer TE cells were lysed within a few
minutes. The dead TE cells could be removed by gently pipetting the
ICMs in a pipette, with a diameter of 40-60 µm.
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Aggregating ICM or Embryo with Tetraploid Embryos
The aggregation plate was prepared as described (Nagy, et al., 2003)
using an aggregation needle. To generate aggregated embryos, the zona
pellucida was removed using acid Tyrode solution. One to three four-cell
stage tetraploid embryos were placed into each of the depressions in the
aggregation plate. One cloned eight-cell stage embryo, one cloned ICM
or one fertilized ICM was placed into one depression. To improve the
aggregation efficiency, embryos in each depression should be physically
attached. The aggregates were cultured in a humidified incubator with 5%
CO2 at 37 ºC for 24-36 hours to reach the blastocyst stage. Successfully
aggregated embryos were transferred into the uterine horns of
2.5-day-postcoitum (dpc) pseudopregnant ICR females mated with
vasectomized males. On Day 19.5 of gestation, the recipient females were
subjected to cesarean section, and live pups were nursed by lactating ICR
females.
Immunostaining
The blastocysts were fixed in PBS supplemented with 4%
paraformaldehyde for 15 min at room temperature (RT). The blastocysts
were then permeabilized using 0.1% Triton X-100 in PBS for 15 min at
RT and blocked for 30 min in 3% BSA in PBS. All primary antibodies
against Oct3/4 (sc-5279, Santa Cruz) and Cdx2 (MU392A, Biogenex)
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were diluted in the same blocking buffer and incubated with the samples
overnight at 4 ºC. The blastocysts were treated with a fluorescently
coupled secondary antibody and then incubated for 1 h at RT. The nuclei
were stained with Hoechst 33342 (Sigma) for 3 min at RT.
The placentae and fetuses were fixed in 4% paraformaldehyde, frozen in
Tissue-Freezing medium, sectioned at a thickness of 10 microns. The
sections were stained with primary rabbit antibody against EGFP (MBL,
1:1000) for one hour at 37 ºC. After rinsing, sections were incubated for
one hour at 37 ºC with a secondary goat anti-rabbit FITC-labled antibody
(Jackson, 1:500). The nuclei were counterstained with Hoechst 33342.
Simple Sequence Length Polymorphism (SSLP)
Genomic DNA was isolated from the fetuses with Phenol: Chloroform:
Isoamyl alcohol (25:24:1) using standard protocol. The simple sequence
length polymorphism (SSLP) analysis were performed for D1Mit46、
D2Mit102 and D6Mit15 using PCR. PCR reaction mixtures were
denatured at 94°C for 5 minutes and cycled at 94°C for 30 seconds, 55℃
~60°C for 30 seconds, and 72°C for 30 seconds followed by final
extension at 72°C for 10 minutes after completion of 40~45 cycles. The
PCR primers sequences for SSLP were obtained from the Mouse Genome
Informatics website (The Jackson Laboratory,
http://www.informatics.jax.org).
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Quantitative RT-PCR
Total RNAs were extracted from placentae and organs using Trizol
Reagent (Invitrogen), following homogenization by power homogenizer.
cDNA was synthesized from 1μg of total RNA using ReverTra Ace First
Strand cDNA Synthesis kit (Toyobo) with oligo dT as a primer. Gene
expression levels were measured by BioRad CFX96 system using SYBR
Green Realtime PCR Mast Mix (Toyobo). PCR reaction mixtures were
denatured at 94°C for 5 minutes and cycled at 94°C 10 seconds, 60°C for
20 seconds, and 72°C for 20 seconds followed by final extension (Melt
Curve detection) at 72°C to 94°C for 10 minutes after completion of 40
cycles. And all the gene expression levels are normalized by β-Actin and
Gapdh in placentae and fetuses, respectively. Primers used for
quantitations are chosen from PrimeBank (pga.mgh.harvard.edu) and
listed in Supplemental Table 1 (Inoue et al., 2002; Ogawa et al., 2006;
Singh et al., 2006; Naidu et al., 2010).
Statistical Analysis
We compared the efficiency of birth rates between groups using
Chi-square test. Differences of gene expression levels between groups
were analyzed by means of Student t-test. All statistical analyses were
done applying SPSS software 13.0.
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Supplemental References
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Tanemura, K., Kaneko-Ishino, T., Ishino, F., and Ogura, A. (2002).
Science 295, 297.
Kishigami, S., Mizutani, E., Ohta, H., Hikichi, T., Van Thuan, N.,
Wakayama, S., Bui, H.T., and Wakayama, T. (2006). Biochem. Bioph.
Res. Co. 340, 183-189.
Li, J., Ishii, T., Feinstein, P., Mombaerts, P. (2004). Nature 428, 393-399.
Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. (2003).
New York, Cold Spring Harbor Laboratory Press, pp. 483-484.
Naidu, S., Peterson, M.L., Spear, B.T. (2010). Gene 499, 95-102.
Ogawa, H., Wu, Q., Komiyama, J., Obata, Y, and Kono, T. (2006). FEBS
Lett. 580, 5377-5384.
Singh, U., Kalinina, E., Konno, T., Sun, T., Ohta, H., Wakayama, T.,
Soares, M.J., Hemberger, M., and Fundele, R.H. (2006). Differentiation
74, 648-660.
Solter, D., and Knowles, B.B. (1975). Proc. Natl. Acad. Sci. USA 72,
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Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson, K.R., Yanagimachi,
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