Gene Expression Profile Normalization in Cloned Mice by Trichostatin ...

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Takashi Kohda,1 Satoshi Kishigami,2,* Tomoko Kaneko-Ishino,3. Teruhiko Wakayama,2 and Fumitoshi Ishino1. Abstract. Cloning mammals by somatic cell ...
CELLULAR REPROGRAMMING Volume 14, Number 1, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/cell.2011.0062

Gene Expression Profile Normalization in Cloned Mice by Trichostatin A Treatment Takashi Kohda,1 Satoshi Kishigami,2,* Tomoko Kaneko-Ishino,3 Teruhiko Wakayama,2 and Fumitoshi Ishino1

Abstract

Cloning mammals by somatic cell nuclear transfer (SCNT) has become an established procedure, but the success rate remains low and gene expression abnormalities are also observed. In addition, SCNT pups exhibited an abnormal gene expression profile with a high degree of heterogeneity among individuals. Recently, we reported that somatic clones treated with trichostatin A (TSA) exhibited a significantly improved success rate, probably due to its effects on chromatin remodeling and histone modification in early embryos. Here we show that the TSA treatment also improves the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up- or downregulation in the TSA clone pups decreased to half of the conventional SCNT pups, and the variation among individuals observed in the SCNT pups was also reduced to the level of the pups produced by the intracytoplasmic sperm injection (ICSI) method. Interestingly, the total gene expression profile of the TSA clones came to resemble that of the ICSI pups.

long-term transcriptional regulation has still not been determined. Previously, we demonstrated that, despite their normal appearance, somatic cell-cloned mice exhibited a significantly abnormal gene expression profile in neonatal tissues and that the genes dysregulated in the clones by SCNT varies among the cloned individuals (Kohda et al., 2005). Therefore, to elucidate the long-term effects of TSA treatment on transcriptome regulation in somatic cell cloning, we examined the gene expression profile in the neonatal tissues of TSA clone mice using a DNA microarray method.

Introduction

T

he success of somatic cell cloning has demonstrated that the somatic cell nuclei can be reprogrammed so as to acquire totipotency. This technique has the distinct characteristic of reprogramming over the whole range of the establishment of induced pluripotent stem (iPS) cells, because somatic cells can be directly reprogrammed to the totipotent state of the zygote in the course of cloning, during which time they are gradually reprogrammed to take on the pluripotent embryonic stem cell-like state in the process of iPS cell establishment. However, the reprogramming molecular mechanism in the somatic cell nuclear transfer (SCNT) procedure has not been elucidated and the somatic cell cloning success rate remains very low. We have reported that the treatment of SCNT embryo with trichostatin A (TSA) results in a significant improvement in the success rate (Kishigami et al., 2006, 2007). Subsequently, significant improvement in cloning efficiency resulting from TSA treatment has also reported in other mammals, such as porcine, bovine, and rabbit models (Iager et al., 2008; Li et al., 2008; Shi et al., 2008). Recently, we reported that TSA enhances the reprogramming of somatic nuclei chromatin remodeling and histone modification in two-cell stage embryos (Bui et al., 2010), whereas the effect on

Materials and Methods Ethics statement. All procedures described here were reviewed and approved by the Animal Experimentation Committee at RIKEN and were performed in accordance with the RIKEN Guiding Principles for the Care and Use of Laboratory Animals. Mice All mice analyzed in this study were produced by genetically identical crosses, for example, C57BL/6 · DBA/2 (BDF1). All neonatal mice were delivered by Cesarean

1

Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. Center for Developmental Biology, RIKEN Kobe Institute, Kobe, Japan. 3 School of Health Sciences, Tokai University, Kanagawa, Japan. *Present address: Graduate School of Biology-Oriented Science and Technology, Kinki University. 2

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46 section. After confirmation of the start of respiration at the time of Cesarean section, the pups were sacrificed, and then the liver and brain were collected and immediately frozen in liquid nitrogen and stored at - 80C until use. Somatic cell cloning Cumulus cell donor nuclear transfer was performed as described elsewhere (Wakayama et al., 1998). TSA treatment was applied as described previously (Kishigami et al., 2006, 2007). Briefly, 50 nM TSA was added to the activation media for 6 h and then the cells were washed and transferred to KSOM containing 50 nM TSA for 4 h. Following complete washing, zygotes were cultured so as to permit development. Each group of embryos was transferred into pseudopregnant female mice. The mice conceived by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) used as the control were produced in the same way as previously described (Bui et al., 2010).

KOHDA ET AL. RNA preparation Total RNA was prepared from neonatal tissues using ISOGEN (Nippon Gene Co. Ltd., Tokyo, Japan), as described previously (Kohda et al., 2005). Total RNA was further purified by the RNeasy Mini kit according to the manufacturer’s protocol (Qiagen, Chatsworth, CA, USA). DNA microarray Analyses using the DNA microarray were performed with an Agilent system (G4122F; Whole Genome (4 · 44K) Oligo Microarray). The probes for the microarray were prepared and labeled by Cy3 according to the manufacturer’s protocol (Agilent Technologies, Englewood, CO, USA). Arrays were scanned with a G2565BA Microarray Scanner System (Agilent Technologies). The expression levels of the different genes were assessed using ‘‘Feature Extract (Agilent Technologies).’’ The signals were normalized using the qspline algorithm implemented in the Bioconductor package of the

FIG. 1. Gene expression in the mice conceived by conventional SCNT or SCNT with TSA treatment. The intensity of each signal from the DNA microarray was normalized by dividing it by the mean signal intensity of the IVF samples. If the value exceeded 1, it was used as the fold increase; if the value was less than 1, the inverse was used as the fold decrease. Genes with similar expression patterns among the ICSI individuals were clustered using Cluster 3.0, and the fold change was plotted in the gene order resulting from the cluster analysis. The gene expression profile of neonatal liver (A–C) and brain (D–F) in four samples of the control IVF (A, D), conventional SCNT (B, E), and SCNT with TSA (C, F) were plotted.

GENE EXPRESSION PROFILE OF SCNT WITH TSA TREATMENT statistics program R. To extract transcripts commonly exhibiting a more than twofold upregulation in the four clone pups, the fold change of each signal in the individuals within one group (SCNT and TSA) was calculated against the average of the IVF control and then the mean difference was checked with the Tukey test ( p < 0.05) using R.

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Results We analyzed the gene expression profile in the neonatal liver of four control pups produced by IVF (Fig. 1A) and four cumulus cell clones (Fig. 1B) using a DNA microarray with 41,174 probes. In the conventional cumulus cell clone

Table 1. Genes Exhibiting Increase or Decrease in SCNT Pup Liver and Normalized in TSA Fold change Gene name U20264 AK087779 AK051522 AK040092 2610027H17Rik AK038045 AK050807 AK050242 NM_001013789 Chka Stra6 AK046412 AK051621 AK084873 Epas1 Tanc1 AK083952 AK076939 AK085204 4930588G05Rik AK033340 AK048657 AK079436 AK079230 AK035046a TC1469196 AK038328a Tns1 Gfod1 Nrip1 AK050555 AK031434 Mtss1 AK047526 AK047848 BC010335 AK078994 AK047019 A630081D01Rika TC1434196 Eml5 Sap30bp Smg6a E130120C16Rika AK048969 AK036907 Centg2 9530091C08Rik 8030498J20Rik AK054507 a

Fold change

SCNT

TSA

Gene name

SCNT

TSA

0.10 0.16 0.16 0.18 0.19 0.19 0.19 0.20 0.20 0.21 0.21 0.21 0.21 0.21 0.21 0.22 0.22 0.22 0.22 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.25 0.25 0.25 0.25 0.25 0.25 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26

0.76 1.05 0.61 0.73 1.08 0.59 0.66 0.54 0.69 0.61 0.97 0.60 0.60 1.11 0.55 0.60 0.70 0.55 1.01 0.98 0.85 0.55 0.70 0.84 0.88 0.77 0.93 0.64 0.55 0.50 0.65 0.77 1.11 0.78 0.66 0.56 0.55 1.81 0.55 0.97 2.00 0.55 1.04 0.93 0.53 0.85 0.63 0.81 0.63 0.55

NAP124059-1 Hist1h3aa 1700031C06Rik Calca Serpina6 Ifi27a A_52_P1101503 Rmrp G0s2 Gata3 Sox9 Guca1a Pfkp Tnf Ldhc Oosp1 AV231787 Tnnc1 C130050O18Rik Bmp10 2610035D17Rik Smpdl3b Olfr74 B230317C12Rik Ahnak Aqp7 AK014119 Tcrg Ksr1 Cst8 2310043J07Rik Mras Vdr Stambpl1 Oosp1 Ccl4 Osm Tbx21 Pdcd1 ENSMUST00000065731 Shbg LOC628211 Lgals1a Aqp7 Mras TC1514546 Hist1h1e 1700088E04Rik Ifitm1 Tdgf1a

5.53 4.96 4.61 4.36 3.99 3.78 3.77 3.73 3.70 3.68 3.67 3.64 3.58 3.30 3.26 3.23 3.22 3.19 3.15 3.15 3.15 3.14 3.12 3.11 3.09 3.07 3.05 3.03 3.02 3.00 2.99 2.98 2.94 2.92 2.91 2.90 2.89 2.88 2.88 2.87 2.85 2.84 2.83 2.82 2.81 2.79 2.76 2.76 2.76 2.75

1.47 0.84 1.80 1.09 0.83 1.43 1.39 1.72 1.74 1.15 1.94 1.74 1.61 1.28 1.15 1.49 1.33 1.31 1.22 1.21 0.75 1.39 1.25 1.01 1.23 1.24 1.25 0.88 1.11 1.56 0.97 1.58 0.79 1.67 0.80 1.41 1.51 1.33 1.84 1.74 1.02 1.00 1.44 1.22 1.70 1.50 1.15 0.98 0.14 0.93

Commonly up- or down-regulated in liver and brain.

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neonate, the gene expression profile was substantially changed. The number of the transcripts commonly exhibiting a more than twofold upregulation in the four clone pups was 885, whereas the number exhibiting down regulation was 1550. At the same time, the number of transcripts exhibiting more than twofold up- or downregulation in at least one of the four pups was 4146 and 4212, respectively.

Next, we analyzed the cumulus cell clones treated with TSA in the same manner (Fig. 1C), and found that the number of the transcripts that commonly exhibited a more than twofold upregulation in the four clone pups was 781 and for twofold downregulation was 612. The genes that exhibited aberrant upregulation in the conventional clones were unchanged in number, whereas the genes exhibiting

Table 2. Genes Exhibiting Increase or Decrease in SCNT and TSA in the Liver Fold change Gene name AK078857 Ptbp2a 2010107C10Rik AK145625 8030488J09Rik Ifrd1 Slc25a25 AK031320 TC1467208a AK045690 Xlr4ba AK048349 Slc25a25 ENSMUST00000094393 AK172662 AK039978 Slc25a25 4631416L12Rik AK030494 Txnip 2610207I05Rik Gadd45b Cry2 AK086877 AK033329 Nfe2l2 Zbtb20 5830410F13Rika TC1507087 Wfikkn1 9430098F02Rik Trim27 5930436O19Rik Crtam D130076A03Rika AK028471 AK085960 Chchd7 Fgd4 AK047137 AK085234 C79248a AK033846 D18Ertd232e E430022E14Rik Erbb2ip Pard3 Arnt TC1541797 AK089858 a

Fold change

SCNT

TSA

Gene name

SCNT

TSA

0.05 0.08 0.09 0.09 0.12 0.12 0.13 0.14 0.15 0.15 0.15 0.16 0.16 0.16 0.18 0.18 0.18 0.18 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.20 0.20 0.20 0.20 0.21 0.21 0.21 0.21 0.21 0.21 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.23 0.27

0.13 0.08 0.03 0.17 0.17 0.18 0.14 0.34 0.09 0.26 0.36 0.19 0.12 0.30 0.47 0.20 0.21 0.18 0.43 0.22 0.45 0.33 0.28 0.30 0.38 0.15 0.23 0.38 0.21 0.18 0.27 0.15 0.36 0.22 0.21 0.44 0.29 0.22 0.25 0.19 0.47 0.26 0.47 0.29 0.22 0.31 0.36 0.48 0.32 0.06

Zfp748 6030458C11Rik 1700065O13Rik 2310047L11Rik D330038O06Rik Chac1 Sh3rf1 Zbtb3 TC1498661 LOC639396 BC050092 TC1434594 Gpr84 5930430L01Rik AK011803 AK052902 4930481A15Rik Zfp418 Zfp84 AK042809 Ptger2 Zfp87 Runx1 Thoc7 TC1410447 AK079452 BB114266 Rag1 Cpn1 A630031M23Rik Pctk2 Dars Eif3s8 R3hdm1 1810047C23Rik AK051618 Taz Rab5b Pde5a AK044799 Clec2d C3ar1 Tnrc15 Kalrn ENSMUST00000014957 AK079215 Zfp11 6030458C11Rik Phka2 5830417I10Rik

7.13 6.58 4.88 4.85 4.79 4.78 4.77 4.71 4.63 4.49 4.32 4.07 4.06 4.01 3.98 3.96 3.86 3.82 3.81 3.68 3.66 3.63 3.56 3.56 3.55 3.49 3.47 3.46 3.33 3.30 3.24 3.21 3.19 3.18 3.17 3.16 3.16 3.14 3.12 3.07 3.03 3.02 3.01 2.99 2.98 2.95 2.92 2.87 2.86 2.83

8.46 7.90 2.51 7.99 3.69 3.52 4.75 2.38 2.05 8.30 4.02 8.96 2.00 7.21 6.37 9.25 2.17 3.90 2.23 3.12 2.87 2.62 2.85 4.79 2.04 3.21 2.66 2.79 5.91 6.24 3.18 4.27 2.72 3.52 2.61 2.25 4.05 3.07 2.26 5.04 2.84 2.06 2.97 3.62 2.10 4.95 2.66 4.90 4.03 2.31

Commonly up- or downregulated in liver and brain.

GENE EXPRESSION PROFILE OF SCNT WITH TSA TREATMENT downregulation in the SCNT clones was reduced by the TSA treatment. The transcripts with and without the normalization resulting from the TSA treatment are summarized in Tables 1 and 2. As previously reported, conventional cumulus clone pups displayed abnormal gene expression, with a wide degree of

A

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variation across the clones. In the case of the TSA-treated clones, the number of the transcripts in at least one of the four pup groups with more than twofold up- or downregulation was 2174 and 1512 in the neonatal liver, respectively. As shown in Figure 2, the standard deviation in the microarray signals among the four pups in each of the

3500

0.06

IVF(liver)

0.05 IVF(brain)

0

2000 1500 0

500

500

1000

Frequency

Frequency 1000

2500

1500

3000

2000

D

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

STDEV/MEAN

B

1.0 1.5 STDEV/MEAN

2.0

2.5

1000

E 1500

0.20

1000

0.28

0

0

Frequency

SCNT(brain)

500

Frequency 500

SCNT(liver)

0.0

0.5

1.0

1.5

2.0

0.0

2.5

0.5

STDEV/MEAN

C

1.0 1.5 STDEV/MEAN

2.0

2.5

1500

F 0.12

TSA(brain)

0.09

0

1000 0

500

500

Frequency

1500

Frequency 1000

2000

TSA(liver)

0.0

0.5

1.0

1.5

STDEV/MEAN

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

STDEV/MEAN

FIG. 2. Gene expression variation in SCNT with and without TSA treatment. Gene expression variation among the individuals in each group is represented as the value for the standard deviation in the microarray signals divided by the mean signal. These values of the liver (A–C) and brain (D–F) for the IVF control (A, D), SCNT (B, E) and SCNT with TSA treatment (C, F) were plotted as histograms (each break was 0.01). The most frequent values are also presented in terms of ‘‘mode.’’

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experimental groups was calculated, and a histogram drawn. The most frequent standard deviation for a conventional clone was 0.2, whereas that for an IVF control was 0.06. The standard deviation peak for a TSA-treated clone was 0.12. This means TSA treatment not only normalize gene expression level but also reduce the variation of gene expression.

The gene expression changes in SCNT and their normalization were also observed in neonatal brain in the same individuals (Fig. 1D–F), but the commonly affected genes in the liver and brain comprised approximately 10% (Tables 3 and 4). The standard deviation in the gene expression was also larger in SCNT in the neonatal brain (mode = 0.05 in IVF, whereas the mode = 0.28 in SCNT), and TSA treatment also

Table 3. Genes Exhibiting Increase or Decrease in SCNT Pup Brain and Normalized in TSA Fold change Gene name AK082373 2900027M19Rik AK038604 AK028486 AK047779 AK042559 AK035046a AK042974 AK042794 AK039039 AK029415 AK043453 AK039932 AK039246 Opcml AK045769 AK032764 AK048842 AK082198 AK080985 AK047123 Cdh10 2900022M07Rik AK086620 Copg2as2 AK090149 AK077975 A830054O04Rik Sorbs2 AK045636 AK047993 9030425P06Rik 6720482D04 AK053189 AK089705 AK048858 A630081D01Rika Gria4 Sox5 AK038647 AK040469 Smg6a 9630039A02Rik AK047015 AK080560 E130120C16Rika A930035E12Rik Arpp21 AK053316 AK038328a a

Fold change

SCNT

TSA

Gene name

SCNT

TSA

0.05 0.05 0.06 0.07 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.11 0.11 0.11 0.11 0.11 0.11 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.13 0.13 0.13

0.79 0.86 1.36 0.61 0.78 0.78 1.20 1.23 1.20 0.73 0.99 1.06 0.55 1.06 0.76 0.84 0.73 0.80 0.59 0.96 0.64 1.05 1.05 0.92 0.78 0.73 0.65 1.06 0.90 1.13 0.91 0.70 0.81 0.62 0.78 0.73 0.69 0.81 0.86 0.76 0.70 1.19 0.82 1.25 1.05 1.13 0.93 1.51 0.72 0.77

Igl-V1 Chi3l3 Tdgf1a Ifi27a D630033O11Rik Hsd3b1 NAP107172-1 1190007F08Rik Cmtm2b Zfp87 BC006965 Ryr1 Pex11c Gata1 Dusp23 Hist1h1b Irf6 Acta2 4930504H06Rik Cd52 Olfr446 Lst1 Olfr60 Hist4h4 Prlpo Tmc1 Hist1h3aa BC019731 Zic4 Olfr1443 1700016G05Rik AK031219 Tgm3 4833421E05Rik Etv3 AY344585 Lemd3 B2m 4930481A15Rik 4933413N12Rik NP063118 2010107E04Rik Spink2 Lgals1a AK040677 Gng13 1110054P19Rik 1600014C23Rik 4930572J05Rik 2900053A13Rik

17.00 8.18 7.06 7.01 6.74 6.65 6.53 6.21 6.21 6.16 6.04 5.91 5.76 5.42 5.38 5.37 5.36 5.36 5.14 5.02 4.98 4.95 4.91 4.86 4.77 4.69 4.61 4.57 4.52 4.36 4.36 4.36 4.31 4.29 4.21 4.17 4.14 4.09 4.08 4.07 4.06 4.05 4.04 4.03 4.03 4.02 4.00 3.91 3.85 3.83

0.93 1.25 1.94 1.44 1.53 1.63 1.70 1.59 1.75 1.66 1.92 0.70 1.53 1.76 1.62 0.78 1.08 1.22 1.23 1.12 1.96 1.98 1.87 0.66 1.03 1.59 0.57 1.89 1.65 1.58 1.64 1.86 1.90 0.85 1.45 0.84 1.50 1.85 1.44 1.31 1.85 1.71 1.32 1.43 1.92 1.54 1.84 1.18 0.89 1.06

Commonly up- or downregulated in brain and liver.

GENE EXPRESSION PROFILE OF SCNT WITH TSA TREATMENT

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Table 4. Genes Exhibiting Increase or Decrease in SCNT and TSA in the Brain Fold change Gene name AK047096 AK053534 AK034993 AK043296 Xlr4ba AK048863 AK045894 Fbxl5 TC1479736 Cntn4 AK080684 TC1413093 Xlr3b AK042781 5830410F13Rika Xpo1 AK085907 Dhx9 AK043796 AK085847 8430419K02Rik Ptbp2a LOC664628 AK036012 AK044191 AK037089 TC1467208a C79248a Grid2 4930546H06Rik D130076A03Rika 4732465E10Rik 3110068A07Rik AK038629 2310035P21Rik Prim2 C030038I04Rik Skiv2l2 AK029965 AK047189 AK033876 AK043448 AK086440 2810055G20Rik Zfp91 4831440D22Rik Taf15 D230015P20Rik Pcdhgb4 AK037099 a

Fold change

SCNT

TSA

Gene name

SCNT

TSA

0.05 0.06 0.06 0.06 0.06 0.07 0.08 0.09 0.09 0.09 0.09 0.10 0.10 0.10 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.12 0.12 0.12 0.13 0.13 0.13 0.14 0.14 0.14 0.14 0.14 0.14 0.15 0.15 0.15 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.17

0.31 0.47 0.49 0.22 0.10 0.46 0.31 0.09 0.21 0.46 0.45 0.15 0.10 0.16 0.47 0.30 0.38 0.27 0.43 0.39 0.12 0.03 0.32 0.48 0.26 0.39 0.09 0.16 0.32 0.35 0.18 0.41 0.41 0.48 0.45 0.43 0.35 0.21 0.34 0.44 0.22 0.35 0.44 0.39 0.30 0.50 0.33 0.10 0.27 0.39

Mt4 A_51_P445924 ENSMUST00000081163 AK006604 LOC546361 NAP070138-1 NAP020861-001 AV094648 D5Ertd135e NAP113002-1 Pex1 NAP028543-1 LOC627004 NAP101638-1 0610009B10Rik NAP057018-1 NAP045499-1 Olfr144 AK136065 Smyd1 NAP059572-1 4930544G11Rik LOC544881 4930571C24Rik NAP061671-1 BC036642 4921534H16Rik 4933440N22Rik LOC433882 NAP070792-1 NAP113376-1 NAP028025-1 NAP062835-1 MGC107702 TC1454836 Rps21 A630049P17 NAP028748-1 Tmc6 Gm1499 Il17rd NAP061630-1 ENSMUST00000088062 NAP070880-1 LOC673028 V1ri6 Cyp17a1 RP23-3N21.1 Hes2 Zc3hav1

15.62 14.39 12.26 11.93 11.20 11.13 10.96 10.62 10.58 10.20 10.07 9.66 9.24 9.06 8.70 8.63 8.59 8.57 8.34 8.14 7.79 7.67 7.01 6.96 6.91 6.77 6.76 6.62 6.54 6.42 6.38 5.91 5.87 5.85 5.85 5.81 5.70 5.66 5.58 5.53 5.31 5.30 5.18 5.17 5.17 5.09 5.06 5.02 4.99 4.98

6.42 8.08 7.33 8.39 8.78 6.95 7.40 7.23 9.84 7.71 5.55 7.58 7.12 6.15 4.28 7.43 6.37 4.09 3.93 4.54 6.10 5.67 6.63 4.71 5.38 5.57 3.21 3.43 7.56 4.64 5.37 4.89 5.15 3.75 4.49 4.25 2.50 9.74 3.99 2.95 2.98 3.32 3.07 4.91 3.31 3.01 3.38 5.40 3.74 2.53

Commonly up- or downregulated in brain and liver.

reduced the variation in the gene expression (mode = 0.09 in SCNT with TSA) in the brain (Fig. 2D–F). These data indicated that the TSA treatment effects were not restricted to a single organ. Recently, we reported that the process of ICSI also induces long-term transcriptome perturbations (Kohda et al., 2011). Interestingly, approximately 60 and 20% of the genes that were affected by ICSI treatment in terms of transcriptional

regulation were also commonly affected by somatic cell cloning, with or without TSA treatment, respectively (Table 5). These overlapping findings were statistically significant (by chi-square test, p-value < 2.2 - 16 both for SCNT with and without TSA) and this suggests there are common mechanisms in SCNT and ICSI that may contribute to the observed transcriptome perturbations. We have summarized the numbers of genes affected by SCNT, SCNT with TSA

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KOHDA ET AL. Table 5. Genes Exhibiting Increase or Decrease in SCNT and ICSI in the Liver Fold change

Gene name AK078857 Ptbp2 2010107C10Rik AK145625 8030488J09Rik Ifrd1 Slc25a25 AK031320 TC1467208 AK045690 Ppp1r3g AK048349 Slc25a25 ENSMUST00000094393 AK172662 AK039978 Slc25a25 4631416L12Rik AK030494 Txnip 2610207I05Rik Gadd45b Cry2 AK086877 AK033329 Nfe2l2 Zbtb20 5830410F13Rik TC1507087 Wfikkn1 9430098F02Rik Trim27 5930436O19Rik Crtam D130076A03Rik TC1525352 AK028471 AK085960 4921522E08Rik Chchd7 Fgd4 AK047137 AK085234 C79248 D18Ertd232e E430022E14Rik Erbb2ip Pard3 TC1541797 Lrrc16

Fold change

SCNT

ICSI

Gene name

SCNT

ICSI

0.06 0.10 0.10 0.11 0.15 0.15 0.16 0.17 0.18 0.18 0.19 0.19 0.19 0.20 0.21 0.21 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.24 0.24 0.24 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.26 0.26 0.26 0.26 0.26 0.26 0.27 0.27 0.27 0.27 0.27 0.27

0.14 0.08 0.04 0.18 0.18 0.19 0.14 0.36 0.09 0.28 0.25 0.19 0.12 0.32 0.49 0.22 0.21 0.19 0.44 0.22 0.49 0.33 0.31 0.32 0.38 0.16 0.24 0.40 0.23 0.20 0.29 0.15 0.36 0.23 0.24 0.26 0.46 0.32 0.23 0.24 0.27 0.19 0.50 0.27 0.30 0.22 0.34 0.38 0.33 0.44

Zfp748 6030458C11Rik 1700065O13Rik 2310047L11Rik D330038O06Rik Chac1 Sh3rf1 Zbtb3 TC1498661 LOC639396 BC050092 TC1434594 Gpr84 5930430L01Rik AK011803 AK052902 4930481A15Rik Zfp418 Zfp84 AK042809 Sox9 Ptger2 Zfp87 Pfkp Runx1 Thoc7 TC1410447 AK079452 BB114266 Rag1 Cpn1 A630031M23Rik Pctk2 Dars Il1f9 Eif3s8 R3hdm1 1810047C23Rik AK051618 Taz NAP045909-1 Rab5b Pde5a Ahnak AK044799 Clec2d C3ar1 Tnrc15 Kalrn ENSMUST00000014957

5.70 5.27 3.91 3.88 3.83 3.83 3.81 3.77 3.71 3.59 3.45 3.25 3.25 3.21 3.19 3.17 3.09 3.05 3.05 2.95 2.94 2.93 2.90 2.87 2.85 2.84 2.84 2.79 2.77 2.77 2.67 2.64 2.59 2.57 2.56 2.55 2.54 2.53 2.53 2.52 2.52 2.51 2.50 2.47 2.45 2.42 2.42 2.41 2.39 2.38

7.84 8.46 3.28 8.98 3.87 3.04 6.35 2.54 2.20 11.41 4.40 9.84 2.08 6.01 6.61 8.51 2.40 2.77 2.40 3.53 2.03 3.26 2.66 2.62 3.02 3.76 2.08 3.48 2.69 2.41 6.03 7.91 3.23 4.91 2.45 2.75 3.95 2.94 3.08 3.90 2.22 3.44 2.40 2.57 4.61 2.87 2.37 3.04 5.21 2.41

treatment, and ICSI in a Venn diagram (Fig. 3). As mentioned above, the number of commonly affected genes in SCNT pups was reduced in the TSA-treated SCNT pups, and there was a significant number of affected genes in SCNT with TSA that did not overlap with conventional SCNT (i.e., SCNT with TSA treatment-specific genes). However, almost all of the genes affected by the SCNT with TSA treatment overlapped with the genes that exhibited up- or downregulation in at least one of the four conventional SCNT

clones. It is also evident that the affected genes in SCNT with TSA treatment overlapped well with ICSI pups. Discussion In this study, it is demonstrated that TSA treatment in SCNT not only increases the efficiency of cloning, but also improves the long-term transcriptome integrity (i.e., transcriptional regulation in neonatal tissues). Recently, we have

GENE EXPRESSION PROFILE OF SCNT WITH TSA TREATMENT

53

liver up

down 2296

2529

SCNT

SCNT

TSA

TSA 246

57

717

107

1066

161 88 295

372 23

3

75

9

82

1

9 144

ICSI

130

3

ICSI

4

at least one of 4 SCNT pups

at least one of 4 SCNT pups

brain up

down 2174

3317

TSA SCNT

SCNT 648

87

TSA

407 45

11

169

96

1178

238 0 165

ICSI

277

6

308

45

at least one of 4 SCNT pups

7 16

ICSI

360 17

at least one of 4 SCNT pups

FIG. 3. Summary of the number transcripts affected by SCNT or ICSI. The number of microarray probes corresponding to the affected transcripts by SCNT, SCNT with TSA treatment, and ICSI in the neonatal liver and brain are summarized in a Venn diagram. The total number of the microarray probes was 41,174.

reported that TSA treatment enhances the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modification, DNA replication, and transcriptional activity in two-cell stage embryos (Bui et al., 2010). This evidence suggests that the TSA treatment improved the condition of the SCNT embryo nucleus in general at the two-cell stage. The reduction in the variance of the gene expression level in this study in the SCNT pups which resulted from TSA treatment is in good accord with these observations.

Almost all of the genes affected in both SCNT with TSA and ICSI were included in the genes affected in at least one of the four SCNT pups (Fig. 3). In other words, the alteration in the aberrant gene expression induced in the conventional SCNT procedure was significantly reduced to a smaller number of genes by TSA treatment. Furthermore, the gene profile shifted to one resembling that of the ICSI produced pups. The genes affected by SCNT, especially by SCNT with TSA treatment, overlapped with the ICSI affected genes

54

KOHDA ET AL. Table 6. Genes Exhibiting Increase or Decrease in SCNT But Not Affected in ICSI in the Liver Fold change

Gene name U20264 Xlr4b AK087779 AK051522 Xlr3b AK040092 2610027H17Rik AK038045 AK050807 AK050242 NM_001013789 Chka Stra6 AK046412 AK051621 AK084873 Epas1 Tanc1 AK083952 AK076939 AK033846 AK085204 Arnt 4930588G05Rik AK033340 AK048657 AK079436 C530043K16Rik AK079230 AK035046 TC1469196 AK038328 Tns1 Gfod1 Bclaf1 Nrip1 AK050555 AK031434 Mtss1 AK047526 AK047848 BC010335 AK078994 A630081D01Rik TC1434196 Eml5 Sap30bp Smg6 E130120C16Rik AK048969

Fold change

SCNT

ICSI

Gene name

SCNT

ICSI

0.13 0.18 0.20 0.20 0.21 0.22 0.22 0.23 0.23 0.24 0.24 0.25 0.25 0.25 0.25 0.25 0.26 0.26 0.26 0.26 0.27 0.27 0.27 0.27 0.27 0.27 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.29 0.29 0.29 0.29 0.29 0.29 0.30 0.30 0.30 0.30 0.30 0.31 0.31 0.31 0.31

1.04 0.66 0.73 0.65 0.82 0.75 1.10 0.62 0.71 0.55 0.69 0.63 0.98 0.64 0.64 0.94 0.55 0.61 0.73 0.59 0.51 1.10 0.52 1.27 0.88 0.55 0.73 0.51 0.87 0.89 0.83 0.93 0.66 0.56 0.50 0.51 0.66 0.80 1.11 0.80 0.72 0.57 0.56 0.60 1.00 1.27 0.60 1.11 0.95 0.56

NAP124059-1 Hist1h3a Calca Il1b Serpina6 Ifi27 A_52_P1101503 Rmrp G0s2 Gata3 Guca1a Smyd2 Lst1 Ptgfr Mt1 Tnf Ldhc Oosp1 Tnnc1 C130050O18Rik Bmp10 2610035D17Rik Smpdl3b Olfr74 BF228116 B230317C12Rik Atp5k Drr1 B3galt5 Aqp7 Fcer1g AK014119 Tcrg Ksr1 Cst8 TC1498165 Pdik1l Vdr Stambpl1 Ccl4 1700026L06Rik Osm Tbx21 Pdcd1 ENSMUST00000065731 Shbg D430015B01Rik LOC628211 A_52_P1157520 Lgals1

4.42 3.97 3.49 3.42 3.19 3.02 3.02 2.98 2.96 2.94 2.92 2.81 2.73 2.69 2.65 2.64 2.61 2.59 2.55 2.52 2.52 2.52 2.51 2.50 2.49 2.49 2.49 2.48 2.46 2.46 2.44 2.44 2.42 2.42 2.40 2.39 2.36 2.35 2.33 2.32 2.32 2.31 2.31 2.30 2.30 2.28 2.28 2.27 2.26 2.26

1.58 0.87 1.15 2.00 1.06 1.45 1.56 1.76 1.99 1.17 1.86 1.61 1.44 1.55 1.93 1.39 1.00 1.53 1.30 1.27 1.73 0.82 1.55 1.43 1.85 1.12 1.81 1.61 1.57 1.37 1.22 1.38 0.99 1.25 0.99 1.74 1.60 1.04 1.74 0.75 1.21 1.62 1.50 1.51 1.37 1.05 1.46 1.13 1.60 1.25

(Fig. 3). This suggests that there may be common steps and/ or reactions in SCNT and ICSI which induce this subgroup of affected genes. The close overlap of the SCNT- and ICSIaffected genes suggests that as a result of common technical steps or procedures, such as the injection of the nuclei with a glass pipette, bypassing the acrosome reaction at fertilization, there might be a slight difference of chromatin decondensation or calcium oscillation in SCNT and ICSI. It is

important to identify this common event at an early stage of development and refine the technique to overcome the problem. On the other hand, there remains a group of genes affected by SCNT but not by ICSI (Table 6). These genes are purely affected by the ‘‘reprogramming process’’ of SCNT and not by the manipulation of SCNT such as the injection step with a glass pipette. To understand the mechanism of the

GENE EXPRESSION PROFILE OF SCNT WITH TSA TREATMENT reprogramming by nuclear transfer, it is important to identify the first target genes of the ‘‘reprogramming process’’ of SCNT among the SCNT affected genes comparing ICSI and SCNT embryo in preimplantation stages. It is also reported that one of the major causes of SCNT embryo lethality at the preimplantation stage is ectopic expression of the Xist gene and aberrant X chromosome inactivation, and it was demonstrated that targeted disruption of the Xist gene significantly improves the efficiency of SCNT (Inoue et al., 2010). The combination of TSA treatment and suppression of ectopic Xist expression appears to more effectively normalize the long-term transcriptome integrity and success rate of SCNT. Acknowledgments This work was supported by grants from the JSPS (18510168). Author Disclosure Statement The authors declare that no conflicting financial interests exist.

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Kishigami, S., Mizutani, E., Ohta, H., et al. (2006). Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem. Biophys. Res. Commun. 340, 183–189. Kishigami, S., Bui, H.T., Wakayama, S., et al. (2007). Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer. J. Reprod. Dev. 53, 165–170. Kohda, T., Inoue, K., Ogonuki, N., et al. (2005). Variation in gene expression and aberrantly regulated chromosome regions in cloned mice. Biol. Reprod. 73, 1302–1311. Kohda, T., Ogonuki, N., Inoue, K., et al. (2011). Intracytoplasmic sperm injection induces transcriptome perturbation without any transgenerational effect. Biochem. Biophys. Res. Commun. 410, 282–288. Li, J., Svarcova, O., Villemoes, K., et al. (2008). High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos. Theriogenology 70, 800–808. Shi, L.H., Ai, J.S., Ouyang, Y.C., et al. (2008). Trichostatin A and nuclear reprogramming of cloned rabbit embryos. J. Anim. Sci. 86, 1106–1113. Wakayama, T., Perry, A.C., Zuccotti, M., et al. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374.

References Bui, H.T., Wakayama, S., Kishigami, S., et al. (2010). Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos. Biol. Reprod. 83, 454–463. Iager, A.E., Ragina, N.P., Ross, P.J., et al. (2008). Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos. Cloning Stem Cells, 10, 371–379. Inoue, K., Kohda, T., Sugimoto, M., et al. (2010). Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer. Science 330, 496–499.

Address correspondence to: Fumitoshi Ishino Department of Epigenetics Medical Research Institute Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku Tokyo 113-8510, Japan E-mail: [email protected]