Jaclyn Nguyen
BIOL 509 – Article Summary
Fall 2010
Summary: High-frequency generation of viable mice from engineered bi-maternal embryos
Kawahara, M., Wu, Q., Takahashi, N., Morita, S., Yamada, K., Ito, M., et al. (2007). High-frequency
generation of viable mice from engineered bi-maternal embryos. Nature Biotechnology, 25(9),
1045-1050.
Introduction and Objective
Genomic imprinting is when the allelic expression of a gene depends on whether it came from the
mother or the father. This gene is epigenetically marked on the egg or the sperm and the other copy is
silenced during egg or sperm formation, which is often done by methylation. While few genes depend
on if it came from the mother or father, genomic imprinting places strict limitations on mammalian
development. Characteristic to imprinted genes are differentially methylated regions (DMRs) that
contain a CpG methylation marker specific to the chromosomal parent-of-origin. Kawahara et al. seeked
to examine the role of imprinted genes with the development of bi-maternal mice. Three DMRs that are
known to be methylated during spermatogenesis in mice are located on chromosome 7, 9, and 12. This
article will analyze the influence on two particular regions: H19 and Dlk1-Dio3.
Experiments and Results
Oocytes were designed containing two sets of haploid genomes: one from a nongrowing (ng) oocyte and
the other from a fully grown (fg) oocyte. The ng oocyte is considered “imprint free” whereas the fg
oocyte genome has maternal methylation imprints. Ng oocytes were taken from double-knockout mice
that had deletions in both H19-DMR (Δch7) and Dlk1-Dio3 intergenic germline-derived (IG)-DMR
(Δch12). Females with Δch7 deletion were mated with males that were heterozygous for Δch12 deletion.
This resulted in double-mutant females that were heterozyous for the Δch7 deletion (Δch7-/+) and
heterzygous for the Δch12 deletion (Δch12+/-). Heterozygous Δch7-/+ and Δch12+/- males were mated
with homozygous Δch7-/- females which created double-mutant females with homozygous Δch7-/- and
heterozygous Δch12+/- deletions. NgΔdouble oocytes with double mutations were taken from females with
genotypes Δch-/+ and Δch+/- or Δch-/- and Δch+/-. Finally, ngΔDouble/fg bi-maternal embryos were
reconstructed by serial nuclear transfer.
After growth and development of blastocysts in surrogate mothers, 42 live pups were recovered and
they were all heterzygous for the double mutation on chromosomes 7 and 12, which means that the
genotype ngΔDouble/fg was the only one able to survive. Thirty-eight pups were successfully nursed by
foster mothers and 27of 38 grew to adults. The mean body weights of the 27 pups (1.19 ± 0.02 g) were
similar to those of the wild-type controls (1.20 ± 0.02) at birth. However, they showed postnatal growth
retardation rates that were greater than wild-type mice (>20%). The other 11 pups had slight growth
retardation at birth (0.90 ± 0.06 g) and died within 3 days. It is believed that growth retarded in
ngΔDouble/fg mice was due to the repression of the transcription of Rasgrf1 which is regulated by paternal
methylation imprinting. Its role is to induce growth hormone secretion from the pituitary.
Phenotypic analyses reported that bi-maternal mice were phenotypically similar to the wild-type
controls. Learning and performance were also analyzed using a rotarod test and a water maze-learning
test. Results indicate that the fitness and learning performance of bi-maternal mice were also similar to
those of the controls. However, mice that had showed postnatal growth retardation had deficits in longterm memory. Phenotypic analyses of ngΔch7/fg and ngΔch12/fg showed differences in the two with the
ngΔch12/fg having smaller body and placental sizes.
Gene expression anaylses were also performed using real-time RT-PCR at E15.5 for Igf2 (another gene
that undergoes genomic imprinting), H19, and genes in the Dlk1 cluster (Dlk1, Gtl2, Mirg, and Dio3).
Northern blotting was done on Rtl1. Igf2 has been known to be inherited from the father. As expected,
bi-maternal mice had exhibited low expression of Igf2. Dlk1 and Rtl1 expression was also repressed in
ngΔch7/fg bi-maternal fetuses. In addition, a microarray analysis was done to compare global change of
gene expression between the genotypes ngΔDouble/fg, ngΔch7/fg, ngΔch12/fg, and wild-type controls. Notable
results from this include confirmation that Igf2 and Dlk1 genes were incorporated into chromosome 7
and 12 gene lists, respectively. Mice with the genotype ngΔch7/fg had shown a 13-fold change compared
to ngΔDouble/fg mice. It is previously known that Mmp13 mRNA transcription is simulated by Igfbp3 whose
mRNA expression is regulated by Igf2. Inactivation of Mmp13 has been shown to lead to abnormal
skeletal growth plate development. Thus, the correction of Igf2 in ngΔDouble/fg can fix the abnormal
skeletal development in mice. Microarray analysis also shows that the Dlk1 gene affects a large network
of other genes. It is also known that in a Dlk-null mutant there will be prenatal growth retardation,
postnatal obesity, blepharophimosis (overall narrow opening for the eyes), and skeletal malformation.
Overall, it was shown that there was a normalization of genes in ngΔDouble/fg mice that were differentially
affected in single mutant ng/fg conseptuses. It was thus speculated that Igf2 and Dlk1 play a
complementary role in development of mice.
Conclusion
It was shown from this experiment that bi-maternal mice can be successfully produced at high
frequency comparable to that of bi-paternal mice. Maternal methylation functions to prevent
parthenogenetic development after the early implantation stage. Development is also inhibited after
mid-gestation when there is no regulation by paternally methylated/maternally unmethylated regions
on chromosomes 7 and 12. These regions must be restored and regulated in order for normal
development. Overall, this study has provided further insight on the role of maternal and paternal
genomes during mammalian development. Equivalent genes and their functions can also be found in the
human genome. Studies such as this could allow greater insight in illnesses involving improper genomic
imprinting during development.