in vivo

Research Unit for
Lymphocyte Cloning
As a physical entity, this Unit has existed
since September 2007. The mission of the
Unit is to provide expertise in reprogramming
of somatic cells to the pluripotent state and
in animal cloning to benefit immunological
research. Analysis of the differentiation of ES
cells derived from immune cell nuclei, as well
as analyzing the immune system of the animals
cloned from immune cells, may provide valuable
insight into the development and function of
the immune system. Additionally, some human
immune system cells, e.g. NKT and HSC, may
be difficult to expand to the quantities necessary
for cell therapy approaches. In principal, these
cells could be converted to the pluripotent state,
propagated to the needed quantities, and then
re-differentiated into the appropriate cell type.
Our Unit will attempt to develop this technology.
Unit leader
Andrei Rybouchkin
Research Scientist : Menzorov Aleksey
Technical Staff
: Sakoda Raul
In our recent research we found that serial
nuclear transfer improves derivation of ES cell
lines from NKT cells of inbred [C57BL /6 (B6)]
mice. Previously NKT derived ES cell lines were
obtained only from hybrid strains of mice, for
example B6/129 F1 (Inoue et al., 2005 Current
Biology 15:1114). Derivation from inbred strains
was a significant challenge, likely due to the
generally low viability of inbred embryos which
exacerbated the already low development of
somatic nuclei-derived clones. This problem was
especially acute for the B6 strain of mice that is
most commonly used in immunological studies.
The early preimplantation embryos of this strain
are very sensitive to in vitro culture conditions
and respond to culture stress by increased levels
of the p53 pro-apoptotic transcription factor and
reduced cell number in the blastocysts (Li et al.,
2007 Biol. Reprod. 76: 362). Indeed, we have
found that NKT derived cloned embryos from
this strain of mice rather successfully develop to
the 4 cell stage (60%), but do not generate good
quality blastocysts (less than 1% in comparison
to 15% for B6D2 F1 NKT cloned embryos), thus
making the derivation of B6 ES cell lines a very
difficult task. As a new approach to this problem,
we used Sendai virus induced fusion to transfer
nuclei of 2 cell stage NKT cloned embryos to
the timed matched cytoplasm of in vivo fertilized
in vivo cultured B6D2F2 embryos ( Liu et al., J
Reprod Dev. 2007, 53(4):785) and obtained
greatly improved development of clones. About
40% of cloned embryos (2 cell stage) obtained
by our serial transfer technique reached the
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