Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 73

Fig. 3.6 Diagram of the methods of using overexpression vectors or silencing shRNA vectors
targeted to DAZL , DAZ , or BOULE to increase or decrease their expression, respectively, in dif-
ferentiating human ES cells. Above is the simplified map showing the p2k7 inducible overexpres-
sion vector and below is the simplified map showing the lentiviral inducible vector pLenti4/
Block-iT DEST used to introduce the shRNAs into human ES cells. Subsequent analysis of
VASA-GFP+ germ cells via FACS reveals the effects of overexpressing or ablating these genes on
the number of germ cells that differentiate in vitro (magnification ×50, bar = 10 mm; images
provided by K. Kee)
To determine the role of these genes in later stages of germ cell development and
meiosis, various combinations of DAZ , DAZL , and BOULE were overexpressed in
differentiating hESCs. The expression of key meiotic genes, formation of meiotic
synaptonemal complexes (SC), and production of haploid gametes were subse-
quently examined at 7 and 14 days of differentiation. Overexpression of DAZL
alone or all three genes together led to an increase in mRNA of the meiotic marker
SCP3 in the female and male lines, respectively. Moreover, meiotic spreads and
SCP3 staining showed that a subset of differentiating germ cells that overexpressed
the three DAZ genes underwent extensive SC formation. Significant numbers of
cells had nuclei with SC formation at the leptotene, zygotene, pachytene, and diplo-
tene stages of meiotic prophase I. Overexpression of BOULE alone promoted
extensive SC formation in the female line, while overexpression of DAZ alone led
to increased SC formation in the male line. Due to the known association of the