Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 72

premature ovarian failure and Turner's syndrome. These studies will interrogate the
molecular mechanisms of reproductive disorders and the genetic requirements of
germ cell development (Dominguez and Reijo Pera, unpublished). The differentia-
tion of germ cells from a variety of iPS cell lines will provide an in vitro cell-based
system to address fundamental questions of normal and aberrant early human
development.

3.5 Genetic Requirements of Making Germ Cells

3.5.1 Key Germ Cell Genes

The germ cell-specific DAZ genes are expressed in diverse organisms including
humans and may play integral roles in human PGC development and gametogen-
esis. However, functional proof of the role of DAZ family genes in humans has not
been directly tested due to several factors: difficulties of studying early human
germ cell development, the unique combinations of DAZ genes present in the
human genome in comparison to model organisms, and deletions of the human
Y chromosome DAZ genes often encompassing neighboring genes. Therefore, in
order to ascertain the role of the DAZ gene family in human PGC formation and
gametogenesis, in recent work we used the VASA-GFP reporter system along with
gene silencing and overexpression technologies in differentiating human ES cells
(Kee et al. 2009 ). To silence or overexpress DAZL , DAZ , and BOULE alone or in
combination, specific shRNA (short hairpin RNA) and overexpression vectors were
employed. The shRNAs targeting the three genes were constructed using the
Block-iT inducible H1 lentiviral system (Fig. 3.6 ). All shRNAs were first intro-
duced into pENTR/H1/TO vectors then transferred into pLenti4/Block-iT-Dest
destination vectors, which were used to transduce hESCs on Matrigel. The overex-
pression vectors were constructed by inserting the EF1a promoter, and DAZL ,
DAZ , or BOULE genes into the p2k7 blas vectors (Fig. 3.6 ). The specificity and effi-
ciency of the silencing and overexpression vectors were tested in 293T cells prior
to experiments with ES cells.
After transduction of hESCs carrying the VASA-GFP reporter with individual
shRNAs it was discovered that the silencing of DAZL had significant effects on
PGC formation—a 50% reduction in the number of GFP-positive PGCs generated
in both XX (H9) and XY (HSF-1) cell lines. In other experiments, silencing of
BOULE reduced the GFP-positive population slightly in the XX line, but not in the
XY line, while silencing of DAZ was virtually ineffective in reducing the number
of GFP-positive cells differentiated from either cell line. Surprisingly, BOULE
overexpression increased the VASA-GFP population to nearly 12%, compared to
4% in control cells in the XX line, but was ineffective in the XY line. As overex-
pression of DAZL and BOULE increased both VASA protein expression and the
quantity of VASA-GFP-positive cells produced, it is likely that these genes are
involved in early human PGC formation and development (Kee et al. 2009 ).