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potential to introduce genes of interest as well as mutated versions in order to do
functional studies during PGC development. TALEN (Transcription Activator-Like
Effector Genome Editing) and CRISPR/Cas9 have been shown to be successful in
generating loss-of-function alleles in zebrafish (Auer and Del Bene 2014 ; Irion
et al. 2014 ). Both, by disruption of coding sequence by indel mutations or by dele-
tion of whole loci from the genome, these approaches represent powerful new tools
for future generations of targeted mutagenesis and DNA integration strategies
(Hwang et al. 2013 ; Hruscha and Schmid 2015 ).
Future directions in genome-edited zebrafish protocols should be to improve
germ line transmission of induced mutations and allelic insertions, high efficient
screening of knock-out and knock-in progeny and the conditional regulation of
manipulated genes. The use of the 3′ UTR region of some germ line genes allows
the specific protection of mRNAs from degradation in germ cells while promoting
their degradation in the soma. This approach could be extremely useful in making a
recombinant Cas9 mRNA that would be expressed exclusively by germ cell precur-
sors in order to improve the rate of germ line transmission. Genetically modified
zebrafish lines will provide powerful tools to conduct conditional gene activation
and inactivation approaches for studies of gene function in PGCs.
8.3 Xenopus
Similar to zebrafish, the germ cell lineage in the frog Xenopus laevis is specified by
the inheritance of maternally formed germ plasm. Ultrastructural characterization of
germ plasm in Xenopus has underscored how similar this aggregate of organelles is
across both vertebrate and invertebrate species. Germ plasm contains dense aggrega-
tion of some 500,000 maternal mitochondria, endoplasmic reticulum, lipid droplets,
and several hundred electron dense “germinal” granules, a hallmark of germ plasm
(Marinos and Billett 1981 ; Kloc et al. 2001 ) (Figs. 8.4 and 8.5). Such similarity is
especially interesting when one considers that germ plasm has been reinvented repeat-
edly during evolution as the inheritance mechanism of germ cell specification appar-
ently derived from the basal inductive mechanism (Extavour and Akam 2003 ).
8.3.1 The Germ Line Life Cycle: Continuity
of the Germ Plasm
In Xenopus, as in zebrafish, germ plasm assembles during the earliest stages of
oogenesis within a structure called the Balbiani body (Bb). Kloc et al. (2004b) have
shown that the developing Bb contains a centriole that likely facilitates microtubule-
based transport of germ plasm components, at least in part, driving the formation of
this body. The formation of the Bb and germ plasm is a lengthy and complex pro-
cess (reviewed in King 2014 ). Studies at the ultrastructural level indicate the first
T. Aguero et al.