Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models
Journal of Human Genetics
Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models
Narumi Uno 0 2 3
? Satoshi Abe 0 2 3
? Mitsuo Oshimura 0 2 3
? Yasuhiro Kazuki 0 2 3
0 Chromosome Engineering Research Center, Tottori University , 86 Nishi-cho, Yonago, Tottori 683-8503 , Japan
1 Yasuhiro Kazuki
2 Trans Chromosomics Inc., 86 Nishi-cho , Yonago, Tottori 683-8503 , Japan
3 Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University , 86 Nishi-cho, Yonago, Tottori 683-8503 , Japan
Chromosome transfer technology, including chromosome modification, enables the introduction of Mb-sized or multiple genes to desired cells or animals. This technology has allowed innovative developments to be made for models of human disease and humanized animals, including Down syndrome model mice and humanized transchromosomic (Tc) immunoglobulin mice. Genome editing techniques are developing rapidly, and permit modifications such as gene knockout and knockin to be performed in various cell lines and animals. This review summarizes chromosome transfer-related technologies and the combined technologies of chromosome transfer and genome editing mainly for the production of cell/animal models of human disease and humanized animal models. Specifically, these include: (1) chromosome modification with genome editing in Chinese hamster ovary cells and mouse A9 cells for efficient transfer to desired cell types; (2) single-nucleotide polymorphism modification in humanized Tc mice with genome editing; and (3) generation of a disease model of Down syndrome-associated hematopoiesis abnormalities by the transfer of human chromosome 21 to normal human embryonic stem cells and the induction of mutation(s) in the endogenous gene(s) with genome editing. These combinations of chromosome transfer and genome editing open up new avenues for drug development and therapy as well as for basic research.
Chromosome transfer technology
Chromosomes can be transferred from donor cells to
recipient cells using several techniques of chromosome transfer.
Different research groups are developing chromosome
transfer techniques involving microcell-mediated
chromosome transfer (MMCT) [
], and the transfection of
chromosomes with liposome carriers .
Narumi Uno and Satoshi Abe contributed equally to this work.
Chinese hamster ovary (CHO), A9, and DT40 cells are
mainly used as donor cells in MMCT (Fig. 1a) because they
are capable of micronucleation with elongated inhibition of
] in response to colcemid [
] or TN16 and
griseofulvin treatment [
]. Microcells are isolated by the
centrifugation of cells forming micronuclei. Those
microcells contains one or a few chromosomes and can be fused
with recipient cells using polyethylene glycol (PEG) [
Because the efficiency of PEG-MMCT is low, virus
proteinmediated MMCT such as MV-MMCT and Retro-MMCT
was developed to increase the efficiency of chromosome
Liposome carriers can also be used to transfer
chromosomes. In this process, naked chromosomes are isolated
from cells that have been induced to undergo mitotic arrest
with colcemid treatment. The chromosomes collected by
sucrose cushion centrifugation can be transfected to
recipient cells using liposome carriers [
]. However, the
efficiency of this method is low, so it is less commonly used for
chromosome transfer than MMCT.
Applications of MMCT and the hurdles of conventional genome engineering technology
MMCT has been applied to various fields of molecular
biology and biotechnology, e.g., chromosome mapping,
functional assays associated with chromosome structure
], and the generation of transchromosomic (Tc) animals
. For example, an A9 human monochromosome library
was used to transfer a target human chromosome via
]. This library includes all human autosomes and
X chromosome, which were deposited in the Japanese
Collection of Research Bioresources Cell Bank [
Chromosome mapping is based on a complementation
], and involves the introduction of a human
chromosome via MMCT to rescue the phenotype of the
model cells, such as cancer cell lines or disease model cells.
This technique has been used for the identification of genes
related to tumor suppression [
], genomic imprinting
], DNA repair [
], metastasis and genomic
instability , telomerase regulation [
mitochondrial disorders [
], and lysosomal storage diseases [
MMCT has also been applied to investigate chromosomal
functions such as kinetochore assembly, telomere function,
and high-order chromosome architecture [
8, 26, 27
animals harboring an extra copy of a human chromosome
have also been generated via MMCT [
], including a
Down syndrome (DS) mouse model [
humanized immunoglobulin mice  and cattle models [
MMCT technologies and their applications are described in
more detail in our previous review [
Although large DNA can be transferred using chromosome
transfer technologies, difficulties remain using conventional
genome engineering technologies for: (1) the efficient
production of knockout (KO) animals for humanization and (2)
the multiple and/or sophisticated genome manipulation
required to improve existing human disease- and humanized
animal-models, and to establish functional cell-based models.
Therefore, the development of more efficient and convenient
genome manipulation technology is urgently needed.
Genome editing techniques involving artificial nucleases
mainly use zinc-finger nucleases [
activator-like effector nucleases [
], and clustered
regularly interspaced short palindromic
repeats/CRISPR-associated protein 9 (CRISPR/Cas9) [
]. These genome editing
technologies allow KO or knock in cultures cells as well as
animals to be produced. Details of the genome editing
technologies are described in other reviews [
we describe the development of previously reported
chromosome transfer-related technologies, and a new generation
of chromosome transfer technology combined with genome
Combination of chromosome engineering and chromosome transfer
Chromosome modification in homologous recombination-proficient chicken DT40 cells
Two decades ago, chromosome modification relied on the
occurrence of random events. Tc mice expressing the human
immunoglobulin gene and genes on human chromosome 21
were generated with human chromosome fragments
randomly obtained via MMCT [
] and X-ray
irradiationmediated MMCT [
], respectively. To perform required
modifications without depending on random events, the
homologous recombination (HR)-proficient chicken DT40
cell line, which has the capacity to undergo MMCT, was
used for targeted gene insertion [
] and telomere-associated
chromosome truncation to induce Mb-sized chromosomal
]. Such targeted chromosome modifications
enable fine design of the chromosome and advanced
applications to be made, e.g., the construction of mammalian
artificial chromosome vectors including human artificial
chromosomes (HACs) and mouse artificial chromosomes
(MACs) (Fig. 1b), which can be used to generate humanized
animals containing desired Mb-sized DNA.
Chromosome modification via genome editing in
CHO and A9 cells
Although chromosome modification using HR-proficient
chicken DT40 cells has enabled various unique
This multistep of chromosome modification and MMCT is a detour. b
Diagram of a new chromosome modification and MMCT strategy. The
CRISPR/Cas9-mediated chromosome modification can be completed
in CHO and A9 cells without the need for homologous recombination
in DT40 cells, so the targeted chromosome can be directly transferred
from CHO and A9 cells to the targeted cells
developments to be made, it first requires the transfer of a
targeted human chromosome to DT40 cells and then
retransfer of the modified chromosome to CHO cells before
transferring it to the desired cell type (Fig. 2a).
Additionally, the donor cell type determines the MMCT efficiency,
which is low when using DT40 cells, except for transfer to
recipient CHO cells. Moreover, repeated MMCT is
laborintensive and time-consuming (Fig. 2a). Chromosome
modification using CHO or A9 cells, but not DT40 cells,
can avoid these problems, because repeated MMCT is not
required. CRISPR/Cas9-mediated HR and
telomereassociated chromosome truncation have recently been
achieved in CHO and A9 cells, and the modified
chromosomes were shown to be transferrable (Fig. 2b) [
Although the HR efficiency in CHO and A9 cells is lower
than that of DT40 cells and chromosome modification
requires negative selection with suicide genes or fluorescent
] to eliminate cells harboring unexpected
insertions of the modification plasmid vector, the CHO/A9
cell method has the advantage that the modified
chromosome can be directly transferred to desired cells.
On the other hand, genome editing technologies are
associated with two potential problems caused by the
offtarget effect [
]. The first occurs on the target chromosome
although CHO and A9 cells only contain a single targeted
human chromosome. It is therefore important to ensure that
an off-target sequence is located on the targeted
chromosome. The second occurs on the host chromosome of CHO
or A9 cells. In this case, it is not necessary to identify an
off-target effect because the modified chromosome will be
transferred to other cells from the host cells. Thus, targeted
cells transferred the modified chromosome avoid the risk of
off-target effects by CRISPR/Cas9. This is a particularly
valuable advantage of chromosome modification via
genome editing in CHO and A9 cells than conventional
genome editing in targeted cells without MMCT.
DT40-mediated chromosome modification techniques
require ~6 months for three MMCT procedures to be
completed, including 1 month for MMCT and cell cloning,
and another month for PCR and chromosome analyses
including fluorescence in situ hybridization analysis. In
contrast, CHO- or A9-mediated chromosome modification
techniques can be completed more quickly (~2 months), so
have been able to reduce the start-up hurdles for
chromosome engineering, including the transfer of Mb-sized
Characteristics of HACs/MACs
HACs have mainly been constructed in two ways, the
bottom-up or top-down method. The bottom-up approach
generates de novo HAC formation by component assembly
in host cells [
]. One such HAC vector (tet-O HAC)
with an artificial centromere sequence is conditionally
removable from host cells, so is particularly useful for gene
function analysis [
]. The top-down approach generates
HACs from a native chromosome by chromosome
engineering technology, including gene-targeting and
telomereassociated chromosome truncation in DT40 cells [
(Fig. 1b). It is also possible to construct an artificial
chromosome from any mammalian chromosome or cell type
using the genome editing techniques described above. Most
bottom-up HACs are circular and their construction relies
on spontaneous assembly. In contrast, all top-down HACs
are linear, precisely engineered, and stably maintained in
host cells. The bi-HAC system utilizing both top-down and
bottom-up HACs, and benefitting from the advantages of
both, was reported previously [
Regarding Tc animal production, the generation of Tc
mice carrying HACs with gene(s) of interest [
been achieved by using MMCT to establish mouse
embryonic stem (ES) cells containing the HACs. If
germline-transmissible chimeric animals cannot be
developed using ES cells, cloning technologies can instead be
applied to generate Tc animals via MMCT to fibroblasts.
This technology was used to generate Tc calves producing
human immunoglobulin [
Although HACs are stable in human cell lines and some
cells and tissues of other species, they have shown variable
retention rates in mouse tissues, being especially low in
mouse hematopoietic cells with high turnovers [
overcome this problem, MACs were generated by the
topdown approach to have the same capacity as HAC vectors
with the additional advantage of highly stable maintenance
in mouse tissues including hematopoietic cells. Therefore,
MACs are more valuable gene delivery vectors for the
production of model mice than HACs [
several groups have developed MACs using different
methods, to our knowledge those derived from native
mouse chromosomes via the top-down approach are the
most stable in mice [
]. In summary, HACs/MACs as
gene delivery vectors can deliver large genomes, and are
stably and independently maintained with defined copy
numbers in host cells, as well as being transferrable to
desired cell lines via MMCT [
Gene-loading techniques for HACs/MACs
Five types of gene cloning system can be utilized with
topdown HACs/MACs: (a) HR-mediated gene insertion; (b)
the Cre/loxP-mediated insertion system; (c) the
Cre/loxPmediated translocation system; (d) the multi-integrase (MI)
system; and (e) the simultaneous or sequential integration of
multiple gene-loading vectors (GLVs) (SIM) system. In
system (a), gene loading with unique sequences in HACs/
MACs via HR in DT40 cells has previously been performed
] (Fig. 3a). The same gene-loading system involving
HR in other cells such as CHO and A9, but not DT40 cells,
was succeeded by genome editing technologies .
Fig. 3 Gene-loading methods
applicable to HACs/MACs. a
recombinationmediated gene insertion for gene
of interest (GOI). b
Cre/loxPmediated circular DNA
insertion. c Cre/loxP-mediated
large genomic DNA fragment
loading by reciprocal
translocation. d The MI system.
Five gene-loading vectors can
theoretically be inserted into the
MI system. e The SIM system.
Three gene-loading vectors can
be inserted simultaneously. The
sequential method inserts
geneloading vectors one by one
indefinitely. Both methods
enable the theoretical indefinite
integration of gene-loading
HACs/MACs generally harbor loxP sites for gene
loading. In system (b), circular vectors such as plasmids, P1
artificial chromosomes (PACs), and bacterial artificial
chromosomes (BACs) were successfully loaded onto
HACs/MACs by the Cre/loxP system for various purposes
including functional analysis, monitoring system
development, and gene therapy [
] (Fig. 3b). This loading
system is particularly suited to when the gene of interest is
covered by one of these circular vectors. In system (c),
Mbsized chromosome fragments such as the human CYP3A
cluster and human dystrophin gene (~2.4 Mb) can be loaded
onto top-down HACs/MACs [
] (Fig. 3c). This system
is suitable when the gene(s) of interest exceeds the delivery
size limit of PACs and BACs. It involves manipulation of
the chromosome containing the genomic region of interest
by telomere-associated truncation and loxP targeting. The
genomic region is eventually loaded on the HACs/MACs
by Cre/loxP-mediated reciprocal translocation cloning in
Previously, the HAC/MAC system has mainly employed
the Cre/loxP system in CHO cells for gene loading as
mentioned above. However, two multiple gene-loading
systems, the MI and SIM systems, have recently been
developed for application in HACs/MACs with loxP sites
In system (d) (the MI system), four serine integrases and
one recombinase (PhiC31, Bxb1, R4, TP901-1, and FLP)
are utilized. Using all recombination sites, five GLVs can
theoretically be inserted into the MI-HAC (Fig. 3d). For the
efficient and convenient production of Tc animals carrying
gene(s) of interest, a MAC carrying multi-integration sites
(MI-MAC) was also constructed [
]. The MI-MAC has
been transferred into mouse ES cells to enable gene(s) of
interest to be directly loaded onto it [
]. The MI-MAC has
also been transferred into HepG2 cells to verify cytotoxicity
levels via luciferase reporter assay [
]. The MI-HAC/MAC
system can be used in various cell types and has
successfully achieved the insertion of large GLVs such as
PACs and BACs and multiple genes into the MI-HAC/
]. Although limitations exist regarding the
available drug-resistant genes for five GLVs loading onto
the MI-HAC/MAC vector, this was solved by reusing the
same drug-resistant genes by disrupting them with genome
editing technology (Honma et al., 2017, unpublished data).
This system is particularly valuable for gene loading when
required number of gene loading is within five times and if
the MI-HAC/MAC has already been transferred to the
desired cell type.
In system (e) (the SIM system), two integrases and two
recombinases (PhiC31, Bxb1, Cre, and FLP) are used. This
system is applicable to HACs/MACs with loxP sites, and
either simultaneous or sequential integration can be
performed (Fig. 3e) [
]. The simultaneous integration system
enables the simultaneous integration of three GLVs into
HACs/MACs and further sequential insertions. The
sequential integration system enables GLV loading one by
one. Both methods theoretically allow the indefinite loading
of GLVs onto HACs/MACs. Selection for gene insertion is
performed by switching on/off hypoxanthine-guanine
phosphoribosyl transferase (HPRT) reconstitution or
neomycin resistance. Thus, gene loading onto HACs/MACs has
to be performed in HPRT-deficient cells. Although gene
loading with the SIM system is restricted in HPRT-deficient
cells, HPRT KO in cells of interest can readily be performed
by genome editing technology leading to direct gene
loading onto HACs/MACs. The SIM system is particularly
suitable when efficient simultaneous multi-gene loading or
unlimited gene loading is required. HACs/MACs with MI
or SIM systems are expected to be employed in a variety of
fields and can be chosen depending on the purpose. Taken
together, the combination of five cloning methods onto
topdown HACs/MACs and genome editing will be useful for
the cloning of desired genes and gene clusters. Regarding
bottom-up HACs, systems (a, b, d, and e) are applicable to
bottom-up HACs with loxP sites. However, bottom-up
HACs cannot be used for reciprocal translocation-mediated
gene cloning in system (c) because they are mostly circular
and contain multiple loxP sites.
Humanized animal model: the CYP3A model mouse
The HAC/MAC system is a powerful tool for producing
humanized animal models harboring multiple or large genes
with physiological expression [
]. As an example, the
humanized CYP3A mouse model was generated using
HAC vectors [
]. CYP3A genes encode enzymes involved
in drug metabolism and are associated with the metabolism
of approximately 50% of commercially available drugs.
However, CYP3A enzymes differ between species, so
animal models such as the mouse and rat do not reflect human
CYP3A-related pharmacokinetics. Therefore, humanized
CYP3A model animals are useful for predicting
CYP3Arelated pharmacokinetics and toxicities of new drugs in
CYP3A genes includingCYP3A4, CYP3A5, CYP3A7, and
CYP3A43 form a cluster on human chromosome 7. It is
difficult to introduce large genomic regions into mice by
conventional technologies, so to produce a humanized
CYP3A mouse model, human chromosome 7 was modified
in DT40 cells by chromosome engineering and the CYP3A
cluster was cloned into a HAC vector (CYP3A-HAC). The
CYP3A-HAC was transferred into mouse ES cells to
produce chimeric mice that transmitted the CYP3A-HAC
through the germline. Further crossing with Cyp3a-KO
mice produced fully humanized CYP3A mice that showed
gender-, tissue-, and developmental stage-specific CYP3A
expression, similar to humans [
]. The humanized
CYP3A mice also recapitulated the CYP3A metabolic
activity observed in humans. Furthermore, whole-embryo
cultures of the humanized CYP3A mouse embryo showed
teratogenic effects of thalidomide that are not seen in
rodents . Thus, humanized CYP3A mice are expected to
be useful for drug screening by predicting CYP3A-related
Such drug metabolism-related genes generally form large
clusters, so chromosome engineering including HAC/MAC
technologies and chromosome transfer are powerful
technologies for producing humanized mice carrying these
genes. Furthermore, other humanized models of the human
immune system or particular genetic diseases are likely to
be produced in the future.
Previously, KO of endogenous orthologous genes was
laborious, especially in ES cells for fully humanized model
]. Gene cluster KO requires the deletion of a
large targeted region, mainly using the Cre/loxP system, so
is labor- and time-intensive as well as expensive because
targeting of two loxP sites and Cre/loxP-mediated
chromosomal deletion must be performed in mouse ES cells,
and chimeric mice formation assays are required at each
step. To overcome the problem of ES cell-based
technologies, genome editing technologies can be utilized to induce
mutations, deletions of single genes, and large genomic
deletions for orthologous gene KO [
]. Now, the
combination of chromosome transfer and genome editing
technology is crucial for the efficient production of fully
humanized mice. Genome editing techniques realize the further
modification of human genomic loci on the MAC to produce
humanized mice carrying specific single-nucleotide polymorphisms or
disease-associated mutations, deletions, or amplifications for a
humanized animal models (Fig. 4) [
]. If genome editing
of KO endogenous genes can be applied to
germlinetransmitted Tc animals, both time and cost for mating Tc
and KO animals will be saved. Additionally, the
combination of HACs/MACs and genome editing can be adapted to
generate humanized model rats which offer several
advantages over mouse models [
Replacement of single-nucleotide polymorphisms in humanized animal models
Genome editing technologies enable the further
modification of genomic sequences in pre-constructed HACs/MACs.
Numerous single-nucleotide polymorphisms (SNPs) of
human drug metabolism-related genes are reported to affect
drug pharmacokinetics [
]. For example, CYP3A5
SNPs have been reported in intron 3: CYP3A5*1(6986A)
and CYP3A5*3(g.6986A>G) [
]. CYP3A5*1 allele
carriers express CYP3A5 mainly in the liver and intestine,
while homozygotes of the CYP3A5*3 allele lack almost all
CYP3A5 protein expression [
Recently, an MAC was utilized to produce humanized
CYP3A mice (CYP3A-MAC mice), and human-like
CYP3A-related drug metabolism was observed in the
CYP3A-MAC mice as well as in the CYP3A-HAC mice
(Kazuki et al., 2017, unpublished data) [
]. However, the
CYP3A5 SNP genotype of humanized CYP3A mice was
CYP3A5*3, and the mice lacked CYP3A5 protein
expression. To produce humanized CYP3A mice that can be
Fig. 5 Graphical summary of a
disease model of aneuploidy
syndrome. Chromosome transfer
techniques enable the generation
of a disease model associated
with aneuploidy via the
following methods: i Transfer of
an additional chromosome in the
same genome background; ii the
reproduction of advanced
mutations on the host genome
for research into associated gene
(s) using genome editing; iii, iv
transfer of chromosomes with
mutations or truncated
chromosomes generated by
genome editing in CHO or
DT40 cells; and v identification
of responsible gene(s) or loci
using HACs/MACs with
Mbregions or gene(s)
utilized to predict the CYP3A5 contribution to drug
metabolism, the CYP3A5 SNP on the CYP3A-MAC was
modified from CYP3A5*3 to CYP3A*1 by CRISPR/Cas9 in
both mouse ES cells carrying the CYP3A-MAC and
fertilized eggs carrying the CYP3A-MAC [
86, 87, 106
Modification with CRISPR/Cas9 was achieved by the
transfection of mouse ES cells carrying the CYP3A-MAC
using double-stranded circular DNA containing a 1 kb
region of homology with the SNP, and pronuclear injection
with 135 bp single-stranded DNA with the SNP as the
donor. The resulting CYP3A5*1 mice showed higher
CYP3A5 protein expression in their liver and intestine
compared with CYP3A5*3 mice, and CYP3A5 was found
to contribute more to metabolism in CYP3A5*1 mice.
These results suggested that the CYP3A5 SNP had been
functionally modified, and that the SNP effect could be
recapitulated in mice because CYP3A5*1 mice showed the
CYP3A5*1 carrier phenotype.
HACs/MACs can deliver entire gene regions with
elements for physiological expression, and can maintain
defined copy numbers, especially single copies, in model
]. Therefore, modified SNPs can be more
efficiently identified in Tc animals than in conventional
genome-edited animals with diploid genomes. The
combination of HAC/MAC systems and genome editing
technologies is valuable for validation of the SNP effect in
humans by producing humanized model animals with
corresponding SNPs. Because genome editing technologies
enable human genes to be efficiently modified on HACs/
MACs, disease-associated mutations-, deletions-, and
amplification-harboring models that reflect individual
patient phenotypes can be generated from humanized
animals (Fig. 4).
In vitro models for aneuploidy and cancer
Chromosome transfer is a powerful technology for
producing aneuploidy syndrome cell models such as trisomy 21,
18, and 13, and cancer-initiating cell models to reflect the
contribution of specific chromosome amplifications to
cancer progression. One of the aneuploidy syndromes, DS,
also known as trisomy 21 (Ts21), is the most common
genetic chromosomal abnormality. Infants with DS suffer
from transient abnormal myelopoiesis (DS-TAM) at a high
frequency, and approximately 20?30% of cases
subsequently develop DS-related acute megakaryoblastic
leukemia (DS-AMKL) [
]. Mutations in GATA1, encoding
the megakaryocyte transcription factor, resulting in the
production of N-terminal truncated GATA1 (GATA1s) is
associated with DS-TAM and DS-AMKL [
combination of GATA1 mutation(s) and constitutive Ts21 is
the most likely cause of DS-TAM, and additional mutations
are thought to result in DS-AMKL [
To understand the mechanism of the progression to TAM
in DS patients, Ts21, GATA1s, and GATA1s/Ts21 human
ES cells were generated by combining chromosome transfer
and genome editing technologies [
] (Fig. 5i, ii). Notably,
all ES cell lines generated were isogenic and genetically
defined with the advantage of chromosome transfer.
Zincfinger nucleases were used for the targeted mutation of
GATA1 exon 2 in human ES cells (WT-ES), and human ES
cells in which both alleles were mutated (GATA1s-ES)
were successfully obtained. Human chromosome 21 was
then transferred via MMCT to WT-ES and GATA1s-ES
cells to generate Ts21-ES and GATA1s/Ts21-ES,
respectively. ES-sac-mediated in vitro hematopoietic
differentiation analyses using the DS models revealed that Ts21 and
the GATA1 mutations synergistically contributed to
hematopoietic abnormalities. Although another group established
isogenic cell lines via the spontaneous loss of chromosome
21 from induced pluripotent stem (iPS) cells derived from a
DS patient, they reported the synergistic interaction of Ts21
and GATA1 mutations mainly from an assessment of
nonisogenic cell lines with a different genotype [
]. It is
crucial to establish isogenic cell lines for disease models
harboring chromosome abnormalities.
The DS models generated by the combination of
chromosome transfer and genome editing technology are useful
for studying how GATA1 mutations associate with the onset
of TAM in DS patients. Recently, genomic profiling
suggested that several mutations contribute to the progression
from DS-TAM to DS-AMKL [
GATA1s/Ts21ES may be used to screen factors involved in DS-AMKL
progression through introducing additional mutations into
GATA1s/Ts21-ES cells by genome editing technologies
(Fig. 5ii). The introduction of chromosome 21 with a partial
deletion or specific gene mutation generated by genome
editing technology into normal ES cells, corresponding
to partial trisomy 21, will help us to screen the region or
gene(s) responsible for the disease phenotype (Fig. 5iii, iv).
Moreover, the transfer of HACs/MACs carrying specific
loci or genes on hChr.21 will enable the identification of the
gene (Fig. 5v).
Genome editing studies validating the contribution of
recurrent mutations to colorectal carcinogenesis suggested
that aneuploidy, as the gain of chromosomes such as 7, 13,
and 20, may be associated with aggressive tumor
]. Direct confirmation of colorectal cancer
exacerbation can be achieved by generating aneuploidy
models by MMCT of such human chromosomes. The
combination of the two technologies will be useful for the
generation of in vitro chromosomal abnormality models
with multiple genetic alterations for the functional analysis
of cancer development.
Conclusions and future perspectives
Chromosome transfer using MMCT is a unique and
powerful technology for the generation of genetically modified
animals for humanization and disease models. The
introduction of Mb-sized genomic loci is technically difficult and
takes several years using conventional
BAC/transgenicbased genetic modification in mouse ES cells [
Genome editing techniques simplify the genetic
modification and expand the applications. Moreover, the
combination of chromosome transfer and genome editing offers a
new generation of chromosome engineering.
Future prospects based on this combination are
summarized in Fig. 6. Sophisticated chromosome engineering
enables specific chromosomes to be monitored, leading to
efficient chromosome sorting and transfer, and complex
chromosome modification for various purposes. For
example, CRISPR/Cas9 may be applicable to improve the
efficiency of MMCT. Isolated microcells are a mixed
population containing targeted and/or host chromosomes.
The chromosome tagging system involving dCas9 [
fused with fluorescence proteins may be useful in purifying
microcells containing a targeted chromosome.
Considering the use of HACs in gene and cell therapy, to
avoid the potential contamination of animal components
and unknown viruses derived from fused microcells, HACs
should be constructed in normal human cells such as iPS
cells, and transferred without using cell lines of other
species such as CHO, A9, or DT40 cells. Additionally, it will
be beneficial to develop novel techniques to perform
MMCT from human cells to targeted human cells such as
patient-derived iPS cells.
The humanization of mice and larger animals such as
cattle, pigs, and cats will be streamlined by efficient
orthologous gene(s) KO using genome editing, and the
generation of various humanized Tc animal models may
promote drug development and therapy [
HACs/MACs with gene(s) of interest are constructed, they
can be transferred to various mammalian cells and
maintained independently without integration into the host
genome. The generation of disease model animals such as
Duchenne muscular dystrophy (DMD) with various
deletions or mutations of the human DMD genome
recapitulating individual human patients supports our understanding
of detailed disease mechanisms resulting in different
In addition to generating synergistic driver mutations by
genome editing, aneuploidy models generated by
chromosome transfer provide an important insight into the
hallmarks of cancer. For basic research, the screening of
potential tumor repressor genes or gene clusters including
noncoding RNAs with chromosome mapping by
chromosome transfer will be promoted by the precise manipulation
of chromosomes through genome editing. Chromosome
mapping technology also ensures physiological gene
expression following single chromosome transfer to identify
Taken together, the benefits of the combination of
chromosome transfer and genome editing not only aid
efficiency and the acceleration of research, but also promote
novel approaches and new perspectives in the field, such as
synthetic biology involving genome writing [
Acknowledgments We thank the graduate students and technical staff
at Tottori University for their technical assistance, and Drs H. Kugoh,
M. Hiratsuka, and T. Ohbayashi for critical discussions. Our studies in
this review were supported in part by The Mochida Memorial
Foundation for Medical and Pharmaceutical Research (to Y.K.), The Naito
Foundation (to Y.K.), The Takeda Science Foundation (to Y.K.), the
Regional Innovation Strategy Support Program from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (to Y.K.
and M.O.), the Funding Program for Next Generation World-Leading
Researchers (NEXT Program) from the Japan Society for the
Promotion of Science (JSPS) (to Y.K.), the Basic Science and Platform
Technology Program for Innovative Biological Medicine from
Japan Agency for Medical Research and Development, AMED
(to Y.K.), and JSPS KAKENHI Grant Number 25221308 (to M.O.),
Grant Number 15H04285 (to Y.K.), and Grant Number 15K19615
Compliance with ethical standards
Conflict of interest Dr. M.O. is CEO of, and a shareholder in, Trans
Chromosomics Inc. The remaining authors declare that they have no
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