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BIOLOGY OF REPRODUCTION (2013) 89(2):24, 1–6

Published online before print 26 June 2013.


DOI 10.1095/biolreprod.113.110098

Mouse Cloning Using a Drop of Peripheral Blood1


Satoshi Kamimura,3,4 Kimiko Inoue,3,4 Narumi Ogonuki,3 Michiko Hirose,3 Mami Oikawa,3,5 Masahiro
Yo,3,4 Osamu Ohara,6 Hiroyuki Miyoshi,3,4 and Atsuo Ogura2,3,4,7
3
RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
4
Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan
5
Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

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6
Kazusa DNA Research Institute, Chiba, Japan
7
The Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan

ABSTRACT been the subject of research in technical improvements to


SCNT [3–5]. Thus, donor cell types have been selected for
Somatic cell nuclear transfer (SCNT) is a unique technology their genomic reprogrammability in terms of the efficiency of
that produces cloned animals from single cells. It is desirable producing live clones. However, from a practical viewpoint,
from a practical viewpoint that donor cells can be collected
ease of access and noninvasiveness at cell collection and
noninvasively and used readily for nuclear transfer. The present
study was undertaken to determine whether peripheral blood
readiness for experimental use should also be key determinants
cells freshly collected from living mice could be used for SCNT. of usable donor cell types. This is especially the case when
We collected a drop of peripheral blood (15–45 ll) from the tail invaluable animals such as the only living individual of a
of a donor. A nucleated cell (leukocyte) suspension was particular genotype is used as the donor. In this context, cells
prepared by lysing the red blood cells. Following SCNT using collected from a small volume of peripheral blood cells are
randomly selected leukocyte nuclei, cloned offspring were born expected to fall into this category. However, as far as we know,
at a 2.8% birth rate. Fluorescence-activated cell sorting revealed there have been very few reports of direct cloning from the
that granulocytes/monocytes and lymphocytes could be roughly nuclei of peripheral blood cells, although tail blood cell nuclei
distinguished by their sizes, the former being significantly larger. contributed to the indirect generation of cloned mice via
We then cloned putative granulocytes/monocytes and lympho- chimeric mouse production using SCNT-derived embryonic
cytes separately and obtained 2.1% and 1.7% birth rates, stem cells [6]. Apart from peripheral blood cells, lymphocytes
respectively (P . 0.05). Because the use of lymphocyte nuclei from lymph nodes [7], bone marrow [8], and liver [9] have
inevitably results in the birth of offspring with DNA rearrange- been used successfully for producing cloned mice directly or
ments, we applied granulocyte/monocyte cloning to two indirectly. The present study examined the feasibility of SCNT
genetically modified strains and two recombinant inbred strains. in mice by using nuclei from peripheral leukocytes collected
Normal-looking offspring were obtained from all four strains from living donor animals.
tested. The present study clearly indicated that genetic copies of
mice could be produced using a drop of peripheral blood from
living donors. This strategy will be applied to the rescue of
MATERIALS AND METHODS
infertile founder animals or a ‘‘last-of-line’’ animal possessing Animals
invaluable genetic resources.
B6D2F1 (C57BL/6 3 DBA/2 hybrid) female mice were purchased from
granulocyte, leukocyte, lymphocyte, mouse, somatic cell nuclear Japan SLC (Shizuoka, Japan) and used as donors for oocytes, cumulus cells,
transfer and peripheral blood cells. For collecting peripheral blood cells, Oct4-green
fluorescent protein (GFP) transgenic female mice (GOF18-DPE-GFP [10]),
INTRODUCTION Dppa3-Venus transgenic female mice, a 129XB6-P female mouse, and a
129XB6-F female mouse were used. The Oct4-GFP strain was derived from
Cloning mammals by somatic cell nuclear transfer (SCNT) backcrossing the original transgenic males with B6D2F1 females for .10
is a promising technology with potential applications in generations in our laboratory and used at 8–45 weeks of age. The Dppa3-
Venus strain was generated by pronuclear DNA injection into B6D2F1 3 B6
regenerative medicine, biological drug production, farming embryos in our laboratory. A male founder mouse was mated with B6
industries, and species conservation [1,2]. Many technical and females, and transgenic females at the F2 and F3 generations were used as
biological factors can affect the outcome of SCNT in terms of donors at 50–80 weeks of age. The 129XB6-P and 129XB6-F strains were
the developmental ability of cloned embryos and the health recombinant inbred strains generated from a hybrid between 129 and C57BL/
status of the offspring [3–5]. Among these factors, the donor 6 strains, and female mice at approximately 8 weeks of age were acquired
cell type is thought to be one of the critical factors and has long from stocks at RIKEN BioResource Center. ICR female mice (CLEA Japan,
Tokyo, Japan), 8–16 weeks old, were used as embryo transfer recipients. The
1
animals were housed under controlled lighting condition (0700–2100 h) and
Supported by a Grant-in-Aid (no. 20062012) from the Ministry of were maintained under specific-pathogen-free conditions. All animal
Education, Culture, Sports, Science and Technology of Japan. experiments were approved by the Animal Experimentation Committee at
2
Correspondence: Atsuo Ogura, RIKEN BioResource Center, 3-1-1, the RIKEN Tsukuba Institute and were performed in accordance with the
Koyadai, Tsukuba, Ibaraki 305-0074, Japan. committee’s guiding principles.
E-mail: ogura@rtc.riken.go.jp

Preparation of Donor Cells


Received: 15 April 2013.
First decision: 5 May 2013. Peripheral blood was collected from the tail vein using a heparinized
Accepted: 7 June 2013. calibrated pipette (45 ll; Drummond Scientific Company, Broomall, PA).
Ó 2013 by the Society for the Study of Reproduction, Inc. Blood was transferred to a 1.5-ml tube containing 10–20 ll of 0.1 M EDTA.
eISSN: 1529-7268 http://www.biolreprod.org Collected peripheral blood was treated with erythrocyte-lysing buffer (155 mM
ISSN: 0006-3363 NH4Cl, 10 mM KHCO3, 2 mM EDTA, pH 7.2) and washed three times by

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KAMIMURA ET AL.

centrifugation (1200 3 g for 5 min). After centrifugation, the pellet containing RESULTS
leukocytes and erythrocyte debris was resuspended in Hepes-buffered
potassium simplex-optimized medium (KSOM) [11]. Then, normal-looking In a preliminary experiment, we tested how much peripheral
leukocytes in the cell suspension were used as nuclear donor cells. To blood was needed for preparing donor cells for SCNT.
distinguish between lymphocytes and other cells (mostly granulocytes), the cell Different amounts of peripheral blood were collected from
suspension was treated with lymphocyte-specific fluorescein isothiocyanate
(FITC)-conjugated, pure Phytolacca americana-derived lectin (PWM; EY the tail through an incision made by a blade (Fig. 1A). After
Laboratories, Inc., San Mateo, CA) and was examined using a fluorescence lysing the red blood cells (Fig. 1B) and washing by
microscope or by fluorescence-activated cell sorting (FACS) analysis. We also centrifugation, we placed the remaining cells (mostly leuko-
tried to separate specific leukocyte types by their sizes. To determine the cytes) in a 3- to 4-ll drop of 10% polyvinylpyrrolidone in
accuracy of such size-dependent separation, cells selected using an inverted Hepes-buffered KSOM, according to our routine SCNT
microscope were placed on a glass slide and compressed on the surface using a
CytoSpin III cytocentrifuge (Thermo Fisher Scientific Inc., Waltham, MA) at
protocol. For efficient pickup of donor cells using an injection
600 rpm for 2 min. The cells were stained with May-Grünwald Giemsa stain to pipette, at least 5–10 donor cells needed to be observed in the
identify leukocyte types. visual field at 2003 magnification (the combination of a 203

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objective lens and a 103 eyepiece). We finally determined that
Nuclear Transfer cells from an initial blood volume of approximately 10–15 ll
were sufficient for SCNT. Therefore, in subsequent experi-
Nuclear transfer was carried out as described previously [12,13]. Briefly, ments, we collected approximately 30–45 ll of blood from
B6D2F1 females were superovulated by injection of 7.5 IU of equine
chorionic gonadotropin and 7.5 IU of human chorionic gonadotropin (hCG) at healthy adult mice and a smaller drop (approximately 15–20
a 46- to 52-h interval. At 14–17 h after receiving the hCG injection, the mice ll) from aged or small animals.
were euthanized, cumulus-oocyte complexes were collected from the We first performed SCNT experiments by using donor cells
oviducts, and cumulus cells were removed by suspending them in KSOM selected randomly from a suspension of peripheral leukocytes
containing 0.1% bovine testicular hyaluronidase. Oocytes were enucleated in collected from B6D2F1 female mice. At 24 h after oocyte
Hepes-buffered KSOM containing 7.5 lg/ml cytochalasin B. The donor
nuclei were injected into enucleated oocytes by using a piezo-driven
activation, 51% and 65% of the embryos that survived nuclear
micropipette (Prime Tech Ltd., Ibaraki, Japan). After culture in KSOM for transfer developed to the 2-cell stage in 5 nM TSA and 50 nM
1 h, injected oocytes were incubated in Ca2 þ-free KSOM containing 2.5 mM TSA groups, respectively. These rates were significantly lower
SrCl2, 5 lg/ml cytochalasin B, and 50 nM trichostatin A (TSA; Sigma- than the 2-cell development rate of cumulus cell-derived
Aldrich, St. Louis, MO) under 5% CO2 in air at 378C for 1 h. Next, the embryos (Table 1). This difference was caused mainly by the
oocytes were incubated in KSOM containing 5 lg/ml cytochalasin B and 50 high incidence of fragmentation of leukocyte-derived embryos
nM TSA for 5 h, followed by incubation in KSOM containing 50 nM TSA
alone for 2 h. After being washed, the oocytes were cultured in KSOM under before the first cleavage. After the transfer of 2-cell embryos
5% CO2 in air at 378C. Cloned embryos that had reached the 2-cell or 4-cell into recipient females, 3.6% and 2.0% of them developed into
stage after 24 h or 48 h in culture, respectively, were transferred into the offspring at term following treatment with 5 and 50 nM TSA,
oviducts of pseudopregnant ICR female mice at 0.5 day postcopulation (dpc; respectively (Table 1). Although these birthrates were
the day following sterile mating with a vasectomized male mouse). The significantly lower than those of cumulus cell-derived cloned
pregnant females were euthanized on 19.5 days postcopulation and examined
for fetuses and placentas in their uteri. In the last experiment using a Dppa3-
embryos, our data indicated that peripheral leukocytes could
Venus female mouse, we used a gene knockout strategy with short interfering indeed be used as donor cells for producing living cloned mice.
RNA (siRNA) against Xist. Reconstructed oocytes at approximately 6–7 h Two leukocyte nucleus-derived clones that were nursed by a
after activation were injected with siRNA (25 lM) against Xist using a piezo- foster mother grew into fertile adults and lived until 14 months
driven micropipette, as described previously [14]. and .16 months, respectively (Supplemental Fig. S1; all
Supplemental Data are available online at www.biolreprod.
Fluorescence-Activated Cell Sorting org).
After red blood cells were lysed, peripheral blood cells were stained with We sought to find a method with which to select leukocytes
FITC-conjugated PWM lectin and phycoerythrin-conjugated anti-Mac-1, anti- other than lymphocytes for SCNT, because mice born from
Gr-1, anti-B220, anti-CD4, or anti-CD8 antibody (eBioscience, San Diego, lymphocyte nuclei would have rearranged DNA sequences
CA). The stained cells were analyzed and sorted using a FACSAria instrument [7,9] and thus would not be suitable donor animals from which
(BD Biosciences, San Jose, CA). to make clones. First, we examined whether lymphocytes could
be identified by FITC-conjugated PWM lectin, which report-
Analysis of Rearranged Immunoglobulin Variable Region edly has specificity to such cells. As expected, only some of the
Genes peripheral leukocytes were positively stained with PWM (Fig.
Recombination of the immunoglobulin heavy chain region was detected as
1C). However, FACS analysis using specific antibodies
described by Rohatgi et al. [15] with slight modifications as follows. The first revealed that T cells but not B cells could be identified by
PCR amplification was carried out using a Multiplex PCR kit (TaKaRa Bio PWM lectin (Supplemental Fig. S2).
Inc., Kyoto, Japan) with a mixture of the ‘‘external’’ VH primers (either of two We then examined whether cell size could be used for
sets for VH1–VH7 and VH8–VH16) and the ‘‘external’’ JH primer as described identifying particular types of leukocytes. We prepared
[15]. To obtain clear results, we next performed nested PCR with a mixture of suspensions of granulocytes, monocytes, B cells, helper T
the ‘‘internal’’ VH primers and the ‘‘internal’’ JH primers under the same PCR
conditions as described for the first PCR. The PCR products were analyzed
cells (CD4 þ), and killer T cells (CD8 þ) by FACS analysis and
using 2% agarose gel electrophoresis. measured their sizes by microscopy. As shown in Figure 1D,
monocytes and granulocytes were significantly larger than
Statistical Analysis lymphocytes (P , 0.05). As far as we observed, mouse
peripheral leukocytes were divided roughly into two popula-
Developmental rates of embryos were analyzed using the chi-square test tions, larger than ;8 lm in diameter and smaller than ;6 lm,
with Yates correction. The size of each type of leukocyte was analyzed by one- which were easily identified using differential interference
way analysis of variance, followed by the Tukey-Kramer procedure for
multiple comparisons. A P value of ,0.05 was considered statistically contrast optics (Fig. 1C). Based on these results, we used cell
significant. populations with a diameter of .8 lm as putative granulo-
cytes/monocytes in subsequent SCNT experiments. When
these larger cells were selected and stained with May-
Grünwald Giemsa stain, the majority (85%, 84 of 99) of cells
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MOUSE CLONING USING PERIPHERAL BLOOD CELLS

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FIG. 1. Preparation of donor leukocytes from peripheral blood, and characterization of the donor cells used for SCNT. A) Collection of peripheral blood
from the tail. A small incision was made at the tip of the tail, and approximately 15–45 ll of blood was collected from the incision by using a capillary
tube. B) Peripheral blood cells after treatment with erythrocyte-lysing buffer. Intact cells in the suspension were mostly leukocytes. Bar ¼ 10 lm. C)
Isolated leukocytes stained with FITC-conjugated PWM lectin. FACS analysis revealed that the positively stained cells were T cells but not B cells.
Nonfluorescent cells (upper) were relatively larger than fluorescent cells (lower) (see D). Bar ¼ 10 lm. D) The mean 6 SEM size of each leukocyte type
was isolated by FACS. Diameters were measured on photomicrographs. Bars with different characters are significantly different (P , 0.05). Monocytes and
granulocytes were significantly larger than lymphocytes. E) Putative granulocytes were selected for SCNT, using an inverted microscope, based on their
larger sizes. After they were settled on the surface of the slide glass by centrifugation, they were stained with May-Grünwald Giemsa stain. Therefore, the
cells in this figure are larger than those in B and C. Most cells in the image are granulocytes with a lobular nucleus, but two show the typical features of
lymphocytes with a round nucleus and a high nucleocytoplasmic ratio (arrows). A large monocyte-like cell is also seen in this figure (arrowhead). Bar ¼ 10
lm.

were identified as granulocytes or monocytes (Fig. 1E). As In the next series of experiments, we undertook SCNT using
monocytes were apparently less frequent than granulocytes nuclei from putative granulocytes and lymphocytes to compare
(approximately 1:4 ratio [Fig. 1E]), ‘‘granulocyte/monocyte the developmental potential of their resultant cloned embryos.
cloning’’ will be referred to simply as ‘‘granulocyte cloning’’ At 24 h, granulocyte-derived cloned embryos showed a lower
hereafter. When smaller cells were picked and stained, 91% (32 2-cell formation rate than lymphocyte-derived embryos
of 35) were identified as lymphocytes with a round nucleus and because of the high incidence of fragmentation (22.6% vs.
a high nucleocytoplasmic ratio (data not shown). 9.2%; P , 1 3 106) (Table 2). There was no statistically
3 Article 24
KAMIMURA ET AL.

TABLE 1. Development in vitro and in vivo of embryos cloned from the nuclei of peripheral leukocytes or cumulus cells.*

No. of embryos at term (%)z


No. of embryos at 24 h (%)z
TSA No. of embryos No. of embryos Development Placenta
Donor cell type treatment  cultured 1-Cell Fragmented 2-Cell transferred Implantation to offspring only

Peripheral leukocyte 5 nM 306 50 (16.3) 101 (33.0)a 155 (50.7)a 138 28 (20.3)a 5 (3.6) 0
50 nM 164 13 (7.9) 44 (26.8)a 107 (65.2)b 98 26 (26.5)a 2 (2.0) 0
Cumulus cell 50 nM 181 6 (3.5) 6 (3.5)b 169 (93.4)c 169 66 (39.1)b 13 (7.7) 4 (2.4)
* All donor cells were collected fresh from B6D2F1 females.
 
TSA, trichostatin A.
z
Values with different superscripts within the same column are significantly different (P , 0.05; chi-square test with Yates correction).

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significant difference between lymphocyte-derived embryos We immediately shifted to granulocyte cloning by collecting
and cumulus cell-derived embryos in fragmentation rate. To blood cells from the carcasses. After transfer of the
determine the genomic reprogrammability of these donor cell reconstructed embryos, we obtained one cloned offspring from
types, we cultured embryos for a further 48 h and observed the each strain. As shown in Table 3, there was no significant
rate of development to the 4-cell stage. The 4-cell formation difference in each developmental parameter among the four
rate was significantly higher in the granulocyte-derived strains used, although their genetic backgrounds differed.
embryos than in the lymphocyte-derived embryos (77.7% vs. Finally, we performed a detailed morphological observation
67.5%, respectively; P , 0.005) (Table 2). Among the three of granulocyte-derived embryos at different timings after
experimental groups, cumulus cell-derived embryos showed activation with the aim of detecting the probable cause of
the best 4-cell formation rate (89.1%) (Table 2). After we frequent fragmentation before the first mitosis. The possible
performed embryo transfer, there was no significant difference causes might have been incomplete breakdown of the donor
in the birth rate between the granulocyte and lymphocyte clone plasma membrane at nuclear transfer [16] or some harmful
groups (Table 2). After pups were recovered, three from each effect of the granulocyte secretory vesicles to the recipient
group were fostered by lactating mothers. Two of the oocytes. One or two granulocytes were injected into enucleated
granulocyte-derived clones grew into normal-looking adults oocytes and then observed at 4, 8, and 17 h after oocyte
and gave birth to normal litters when mated with adult males. activation. Oocytes injected with two granulocytes showed a
All the lymphocyte-derived clones died before weaning (21 better (pseudo)pronuclear formation rate and a lower fragmen-
days) from unknown causes. These three offspring were tation rate than those injected with one granulocyte (Supple-
confirmed to be derived from B cells by detecting recombi- mental Table S1). This result indicated that the fragmentation
nation of the immunoglobulin heavy chain region (Supple- of granulocyte-derived embryos was most likely caused by
mental Fig. S3). incomplete breakdown of the donor plasma membrane at
As shown above, the genomes of at least some of the nuclear transfer rather than the harmful effect of the
granulocytes, which had been putatively selected based on size, granulocyte secretory vesicles (see Discussion).
gave rise to normal-looking offspring that grew into fertile
adults. We then applied this granulocyte SCNT technique to DISCUSSION
propagate four mouse strains. The first, the Oct4-GFP strain
[10], was successfully cloned to produce female offspring at a In this study, we used mouse as the model for the study of
birth rate of 1.2% (Table 3). The second strain was Dppa3- SCNT, using peripheral blood cells. Mouse cloning is different
Venus. This strain was originally generated in our laboratory from cloning in farm animals or other large animals in terms of
but accidentally only two aged infertile females remained alive. the protocols for preparing donor cells. In typical SCNT in
Therefore, SCNT was the last means with which to conserve large animals, donor cells such as fibroblasts or epithelial cells
this strain. The first two SCNT experiments failed to produce from biopsy samples are proliferated in vitro and then cultured
term offspring, but one healthy pup and one stillborn pup were under serum-starved or confluent conditions to induce them to
born at the third trial using Xist-siRNA treatment (Table 3). enter the G0/G1 phase of the cell cycle, which is a prerequisite
The third and fourth strains were 129XB6-P and 129XB6-F, for synchrony with the recipient ooplasm [17]. In mice, by
respectively, derived from a set of recombinant inbred strains contrast, donor cells such as cumulus cells [12] or immature
between C57BL/6 and 129 strains. Initially, we attempted to Sertoli cells [18], which are predominantly at the G0/G1 stage
generate cloned mice from cumulus cells of 129XB6-P and by nature, are collected fresh from donor animals and used
129XB6-F female mice for a different project, but the donor immediately for SCNT. This arises at least in part from the
females failed to ovulate on the day of the SCNT experiment. unique character of mouse cells, which are prone to genetic and

TABLE 2. Development in vitro and in vivo of embryos cloned from the nuclei of granulocytes/monocytes, lymphocytes, and cumulus cells.* 
No. of embryos (%)
No. of embryos at 24 h (%)z No. of 4-cells
No. of embryos at 48 h No. of embryos Development Placenta
Donor cell type cultured 1-Cell Fragmentation 2-Cell (% per 2-cells)z transferred Implantation to offspring only

Granulocyte/monocyte 527 44 (8.3) 119 (22.6)a 364 (69.1)a 283 (77.7)a 283 112 (39.6) 6 (2.1) 5 (1.8)
Lymphocyte 327 5 (1.5) 30 (9.2)b 292 (89.3)b 197 (67.5)b 180 69 (38.3) 3 (1.7) 4 (2.2)
Cumulus cell 90 3 (3.3) 4 (4.4)b 83 (92.2)b 74 (89.1)c 74 32 (43.2) 2 (2.7) 2 (2.7)
* All donor cells were collected fresh from B6D2F1 females.
 
All cloned embryos were treated with 50 nM trichostatin A (TSA).
z
Values with different superscripts within the same column are significantly different (P , 0.05; chi-squared test with Yates correction).

4 Article 24
MOUSE CLONING USING PERIPHERAL BLOOD CELLS

TABLE 3. Development in vitro and in vivo of embryos cloned from granulocytes/monocytes of transgenic mice.*
No. of embryos (%)
No. of embryos at 24 h (%)
No. of embryos No. of embryos Development Placenta
Donor strain cultured 1-Cell Fragmentation 2-Cell transferred Implantation to offspring only

Oct4-GFP 574 41 (7.1) 123 (21.4) 410 (71.4) 247  78 (31.6) 3 (1.2) 3 (1.2)
Dppa3-Venus 362 43 (11.9) 70 (19.3) 249 (68.8) 249 69 (27.7) 2 (0.8)z 2 (0.8)
129XB6-P 103 14 (13.6) 17 (16.5) 72 (69.9) 72 23 (31.9) 1 (1.4) 3 (4.2)
129XB6-F 235 12 (6.7) 56 (31.3) 167 (93.3) 139 41 (29.5) 1 (0.7) 4 (2.9)
* All cloned embryos were treated with 50 nM trichostatin A (TSA).
 
Transferred at the 4-cell stage.
z
These two pups were generated from a Xist knockdown experiment (see text), but one was stillborn.

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epigenetic aberrations under in vitro proliferation conditions method for sorting out lymphocytes from cell suspensions
compared with cultured cells from other species [19]. should be FACS, but this needs large cell numbers. Therefore,
Therefore, it is very common that one or more donor mice in this study, we examined the feasibility of selecting cells by
need to be euthanized in each SCNT experiment. In this their sizes, based on data for each cell type obtained by FACS
respect, our cloning procedure using peripheral blood cells is (Fig. 1D). Because granulocytes and monocytes are signifi-
unique and can be used for generating clones without cantly larger than other cell types, we could select them from
sacrificing the donor animals. We also expect that donor cells the leukocyte suspension at approximately 85% accuracy; thus
could be collected repeatedly from one animal. Indeed, in the minimizing the possibility of selecting lymphocytes. As this is
experiments using the Dppa3-Venus strain, the third trial gave a very simple technique, we expect that it could be reproduced
rise to the first cloned offspring. easily in other laboratories after practice. There was a clear
In recent years, the number of genetically modified mouse technical disadvantage to cloning granulocytes because
strains has been increasing rapidly, and this necessitates a approximately 20% of the reconstructed embryos underwent
demand for the ‘‘rescue’’ of strains with accidental or fragmentation before the first cleavage (Tables 2 and 3). This is
genetically caused infertility. Currently, one of the major approximately twofold higher than that of lymphocyte cloning
strategies for the conservation of mouse genetic resources has and fivefold higher than that of cumulus cell cloning.
been intracytoplasmic sperm injection (ICSI). ICSI in the Fragmentation of reconstructed cloned embryos is mostly
mouse is a very reliable technique for fertilizing an oocyte by caused by the disorganization of microtubule organizing
using a single spermatozoon or even an immature one (e.g., a centers in activated oocytes, which might occur in association
spermatid) [20,21]. Therefore, it is very probable that only one with delayed or incomplete intermingling of the donor nucleus
founder or a ‘‘last-of-line’’ male mouse could be used with the cytoplasm [16]. This seemed to be the case with
successfully for ICSI to propagate a valuable strain. Even granulocyte cloning because the rate of fragmentation was
males at 36 months of age can be used for producing offspring significantly decreased when two granulocytes were injected
by round spermatid injection [22]. However, ICSI can be into single enucleated oocytes (Supplemental Table S1). It is
applied in practice only when the donor animals are male and possible that the plasma membrane of granulocytes is more
they have haploid male germ cells in their testes. Conception resilient than that of other donor cell types. Another possibility
with primary or secondary spermatocytes is also possible, but was the harmful effect of enzymes released from the
these younger spermatogenic cells need additional microma- granulocyte secretory vesicles. It is known that the acrosomal
nipulation steps for constructing diploid embryos [23,24]. enzyme contents from multiple spermatozoa used for ICSI
Conversely, when male donors have no haploid germ cells or causes deformation of the injected mouse oocyte [26]. Our
only female donors are available, SCNT rather than ICSI may experiment suggested that the cell toxicity of granulocyte
offer a better chance for rescuing their genotype. Furthermore, vesicles was an unlikely cause of fragmentation in granulocyte
clones because injection with two granulocytes did not
SCNT is also advantageous over ICSI in animal strains needing
increase, but rather decreased, the fragmentation rate.
conservation but carrying multiple mutant genes or those with
In conclusion, the present study has demonstrated for the
an undefined genetic background. Although the reprogramm-
first time that mice could be cloned using the nuclei of
ability of peripheral leukocytes seemed lower than that of other peripheral blood cells. These cells could be used for cloning
standard donor cells including cumulus cells, this technical immediately after collection and no donor animals need to be
difficulty might be overcome by future technical improve- euthanized. This technique would be applicable for generating
ments, as previously achieved with treatment with histone genetic copies of invaluable strains of mice, which cannot be
deacetylase inhibitors, knockdown by siRNA interference preserved by other assisted reproduction techniques such as
against Xist, or their combination [13,14]. Knockdown against conventional in vitro fertilization or ICSI.
Xist was very effective for cloning male, but not female, mice
as we reported [14,25]. In the present study, it was intriguing
that Dppa3-Venus female clones were first obtained when Xist ACKNOWLEDGMENT
knockdown was employed. It is probable that the use of a We thank Dr. Kazuhiro Sudo and Dr. Haruhiko Koseki for their
higher concentration of siRNA against Xist (25 lM) than usual invaluable discussion on the preparation of leukocytes for the identifica-
(5 lM) might have resulted in the birth of female clones, but tion of cell types and detection of rearranged genes in lymphocyte-derived
clone samples, respectively. The strains used in this study, Oct4-GFP
this possibility needs further study. (GOF18-delta PE-GFP line 48; RBRC00869), 129XB6-P (RBRC01007) and
One of the important points of cloning mice using peripheral 129XB6-F (RBRC01005), were provided by RIKEN BioResource Center,
blood cells is avoiding the use of lymphocytes as donors, with the support of the National BioResource Project of MEXT, Japan.
because cloning from such nuclei would inevitably generate
mice with DNA rearrangements [7,9]. The most reliable
5 Article 24
KAMIMURA ET AL.

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