Advances in Genome Editing Tools
14
Marko E. Horb, Anita Abu-Daya, Marcin Wlizla , Anna Noble,
and Matt Guille
CONTENTS
14.1. Generation of Transgenic Xenopus .......................................................................................................................... 207
14.1.1. Beginnings ................................................................................................................................................ 207
14.1.2. Technology Development ......................................................................................................................... 207
14.1.3. pTransgenesis: Streamlining Transgene Construction .............................................................................. 208
14.2. Uses of Transgenic Xenopus .................................................................................................................................... 209
14.3. Genetics and Gene Editing in Xenopus.................................................................................................................... 210
14.3.1. Beginnings ................................................................................................................................................ 210
14.3.2. X. tropicalis and Forward Genetic Screens ............................................................................................... 210
14.3.3. TILLING ................................................................................................................................................... 211
14.3.4. Insertional Mutagenesis ............................................................................................................................ 211
14.3.5. Reverse Genetics Using dsBreaks ............................................................................................................. 212
14.4. Future Directions ..................................................................................................................................................... 214
14.5. Conclusions .............................................................................................................................................................. 215
Acknowledgments ................................................................................................................................................................ 216
References ............................................................................................................................................................................ 216
14.1. GENERATION OF TRANSGENIC XENOPUS
14.1.1. BEGINNINGS
Transgenic animals carry exogenous DNA integrated into
their genome and can be designed to fulfll diverse experimental objectives, from using fuorescent proteins to label specif c
cells/tissues or to observe activity of signaling pathways to using
dominant-negative and constitutively active mutants to disrupt
gene activity. Prior to the development of stable transgenic lines,
experiments to track or disrupt gene function were carried out
via microinjection of RNA, DNA, morpholino oligonucleotides,
or antibodies and through treatments with small molecules, but
these all have related limitations. The mosaic distribution and
progressive dilution with every cell division of microinjected
reagents typically limits their use to the study of genes involved
in early development (Amaya, 2005). Although small molecule
treatments can be applied at any stage of development, care must
be taken that the molecules used are specifc to the genes being
disrupted and that the phenotypes observed are not due to a disruption of a broader range of targets than intended (Vogt et al.,
2011). These experimental limitations can be circumvented
using transgenics. The current transgenic toolset available to
Xenopus researchers permits effcient generation of stable, nonmosaic animal lines and inclusion of elements for temporal control of transgene activity. These allow their use through all stages
of development and make possible the design of transgenic lines
able to disrupt gene activity in a highly specif c manner.
It has been over three decades since the initial reports that
exogenous plasmid DNA was integrated into the Xenopus
genome following its microinjection into fertilized eggs with
successful transmission through the male germline (Etkin
and Pearman, 1987; Etkin and Roberts, 1983; Rusconi and
Schaffner, 1981). However, microinjection of linearized
plasmid DNA is not a practical approach for generation of
stable transgenic lines; it is highly ineffcient, with only 1%
of injected F0 animals showing mosaic integration of the
foreign DNA into their germlines (Yergeau et al., 2010).
Innovation has followed three distinct ways to improve the
transgenic methodologies in Xenopus: f rst, development of
alternative approaches to make genomic integration more
effcient; second, by streamlining the generation of transgenic plasmids carrying the exogenous DNA; and third, by
incorporating elements for temporal regulation of transgene
activity, thus expanding on the versatility of this technology.
This chapter outlines technological advances in Xenopus
transgenesis, followed by an overview of how transgenics
have impacted research using this popular model organism.
14.1.2. TECHNOLOGY DEVELOPMENT
Restriction enzyme-mediate integration (REMI) was the
frst method that made Xenopus transgenesis practical (Kroll
and Amaya, 1996 ). In REMI, transgenic plasmid DNA is
incubated together with isolated sperm nuclei, a restriction enzyme, and Xenopus interphase egg extract and then
injected into mature, unfertilized eggs. One to 16% of the
injected eggs survive past feeding tadpole stages. However,
this is not really a complication, as it is possible to inject
DOI: 10.1201/9781003050230-16
207
14
Marko E. Horb, Anita Abu-Daya, Marcin Wlizla , Anna Noble,
and Matt Guille
CONTENTS
14.1. Generation of Transgenic Xenopus .......................................................................................................................... 207
14.1.1. Beginnings ................................................................................................................................................ 207
14.1.2. Technology Development ......................................................................................................................... 207
14.1.3. pTransgenesis: Streamlining Transgene Construction .............................................................................. 208
14.2. Uses of Transgenic Xenopus .................................................................................................................................... 209
14.3. Genetics and Gene Editing in Xenopus.................................................................................................................... 210
14.3.1. Beginnings ................................................................................................................................................ 210
14.3.2. X. tropicalis and Forward Genetic Screens ............................................................................................... 210
14.3.3. TILLING ................................................................................................................................................... 211
14.3.4. Insertional Mutagenesis ............................................................................................................................ 211
14.3.5. Reverse Genetics Using dsBreaks ............................................................................................................. 212
14.4. Future Directions ..................................................................................................................................................... 214
14.5. Conclusions .............................................................................................................................................................. 215
Acknowledgments ................................................................................................................................................................ 216
References ............................................................................................................................................................................ 216
14.1. GENERATION OF TRANSGENIC XENOPUS
14.1.1. BEGINNINGS
Transgenic animals carry exogenous DNA integrated into
their genome and can be designed to fulfll diverse experimental objectives, from using fuorescent proteins to label specif c
cells/tissues or to observe activity of signaling pathways to using
dominant-negative and constitutively active mutants to disrupt
gene activity. Prior to the development of stable transgenic lines,
experiments to track or disrupt gene function were carried out
via microinjection of RNA, DNA, morpholino oligonucleotides,
or antibodies and through treatments with small molecules, but
these all have related limitations. The mosaic distribution and
progressive dilution with every cell division of microinjected
reagents typically limits their use to the study of genes involved
in early development (Amaya, 2005). Although small molecule
treatments can be applied at any stage of development, care must
be taken that the molecules used are specifc to the genes being
disrupted and that the phenotypes observed are not due to a disruption of a broader range of targets than intended (Vogt et al.,
2011). These experimental limitations can be circumvented
using transgenics. The current transgenic toolset available to
Xenopus researchers permits effcient generation of stable, nonmosaic animal lines and inclusion of elements for temporal control of transgene activity. These allow their use through all stages
of development and make possible the design of transgenic lines
able to disrupt gene activity in a highly specif c manner.
It has been over three decades since the initial reports that
exogenous plasmid DNA was integrated into the Xenopus
genome following its microinjection into fertilized eggs with
successful transmission through the male germline (Etkin
and Pearman, 1987; Etkin and Roberts, 1983; Rusconi and
Schaffner, 1981). However, microinjection of linearized
plasmid DNA is not a practical approach for generation of
stable transgenic lines; it is highly ineffcient, with only 1%
of injected F0 animals showing mosaic integration of the
foreign DNA into their germlines (Yergeau et al., 2010).
Innovation has followed three distinct ways to improve the
transgenic methodologies in Xenopus: f rst, development of
alternative approaches to make genomic integration more
effcient; second, by streamlining the generation of transgenic plasmids carrying the exogenous DNA; and third, by
incorporating elements for temporal regulation of transgene
activity, thus expanding on the versatility of this technology.
This chapter outlines technological advances in Xenopus
transgenesis, followed by an overview of how transgenics
have impacted research using this popular model organism.
14.1.2. TECHNOLOGY DEVELOPMENT
Restriction enzyme-mediate integration (REMI) was the
frst method that made Xenopus transgenesis practical (Kroll
and Amaya, 1996 ). In REMI, transgenic plasmid DNA is
incubated together with isolated sperm nuclei, a restriction enzyme, and Xenopus interphase egg extract and then
injected into mature, unfertilized eggs. One to 16% of the
injected eggs survive past feeding tadpole stages. However,
this is not really a complication, as it is possible to inject
DOI: 10.1201/9781003050230-16
207