Manipulating gene expression precisely using light

Date:

January 24, 2017

Source:

Hokkaido University

Summary:

A scientist has developed a new method to accurately manipulate gene

expression by light illumination and demonstrated its usability by creating doubleheaded zebrafish.

Zebrafish embryos expressing a fluorescent gene after ultraviolet light irradiation. Credit:
Ogasawara S., Duration control of protein expression in vivo by light-mediated reversible
activation of translation. ACS Chemical Biology, Jan 4, 2017.

A Hokkaido University researcher has successfully developed a method to accurately

manipulate gene expression by light illumination and demonstrated its usability by
creating double-headed zebrafish.
It has been difficult to freely manipulate the timing and duration of gene expression
using existing gene manipulation technologies, which depend on organism's gene
regulating mechanism. In recent years, methods using light to regulate gene expression

have been developed, but deemed insufficient to manipulate embryonic development.

This is due to a time lag of several hours that occurs from light irradiation to the
start/cessation of protein production. Existing photocontrol technologies also require
genetic modification, a process that is not only time-consuming but also strictly
regulated by the Cartagena Protocol.
Shinzi Ogasawara of Hokkaido University's Creative Research Institution focused his
research on controlling the process of translating messenger RNA (mRNA) to protein,
instead of the conventional process of transcribing DNA to mRNA. As the new
technology acts on mRNAs, genetic modification is unnecessary. Instead, the mRNAs
were modified in such a way that they could bind to "Initiation Factors (IFs)" when
irradiated with ultraviolet light which then starts the process of translation; whereas
mRNAs were unable to bind to IFs when irradiated with blue light.
To test the new method, fluorescent protein mRNAs were injected into zebrafish
embryos, which were then irradiated with blue or ultraviolet light. The research
confirmed that the embryos illuminated with ultraviolet light produced fluorescent
protein, while those irradiated with blue light showed no existence of fluorescent protein,
indicating that no mRNA translation occurred in the process. The research also found
that protein production starts several minutes after embryos are irradiated with
ultraviolet light and stops several dozens of minutes after blue light irradiation.
Ogasawara succeeded in establishing a technology that considerably shortens the time
lag between light illumination and protein production.
Furthermore, using this new method, Ogasawara created double-headed zebrafish by
accurately controlling the expression duration of squint, a gene that regulates body axis
formation.
Ogasawara says "Our method would be particularly useful to accurately manipulate
embryonic development, and reveal the importance of the timing and duration of gene
expression in biological events. By applying this technology to higher model organisms
such as mice, we hope to help clarify the role each gene plays in the development of
animals as well as in various diseases."

Story Source:
Materials provided by Hokkaido University. Note: Content may be edited for style and
length.

Journal Reference:
1.

Shinzi Ogasawara. Duration Control of Protein Expression in Vivo by LightMediated Reversible Activation of Translation. ACS Chemical Biology, 2017;
DOI: 10.1021/acschembio.6b00684

Rabies viruses reveal wiring in transparent brains:

Researchers use a new method for assessing neural transplant integration
Date:

January 19, 2017

Source:

University of Bonn

Summary:

Scientists have harnessed rabies viruses for assessing the connectivity of

nerve cell transplants: coupled with a green fluorescent protein, the viruses show where
replacement cells engrafted into mouse brains have connected to the host neural
network. A clearing procedure which turns the brain into a 'glass-like state' and light
sheet fluorescence microscopy are used to visualize host-graft connections in a wholebrain preparation.

The surrounding nerve cells in the mouse brain have connected to engrafted neurons. A green fluorescent
rabies virus was used to infect the transplanted neurons (red), from where it spread backwards to
surrounding synaptically connected neurons in the host brain (green). Credit: Dr. Jonas Doerr

Scientists under the leadership of the University of Bonn have harnessed rabies viruses
for assessing the connectivity of nerve cell transplants: coupled with a green fluorescent
protein, the viruses show where replacement cells engrafted into mouse brains

have connected to the host neural network. A clearing procedure which turns the
brain into a 'glass-like state' and light sheet fluorescence microscopy are used to
visualize host-graft connections in a whole-brain preparation. The approach opens
exciting prospects for predicting and optimizing the ability of neural transplants
to functionally integrate into a host nervous system. The results have now been
published in the specialist journal Nature Communications.
Many diseases and injuries result in a loss of nerve cells. Scientists are working on
tackling this challenge by transplanting neurons. In Parkinson's disease, for instance,
this is attempted with implanted dopamine-producing nerve cells. The key question for
such techniques is whether the implanted cells actually connect with the existing neural
network of the host brain and thus compensate the functional loss. "Previous methods
only provided an incomplete or very small-scale insight into the functional integration of
implanted neurons in the brain," says Prof. Oliver Brstle from the Institute of
Reconstructive Neurobiology at the University of Bonn and LIFE & BRAIN GmbH.
Exploiting viral spreading across neurons
Together with scientists of various disciplines at the University of Bonn and cooperation
partners from Cologne and Chicago (USA), the team led by Prof. Brstle developed a
new technique: "This enables the connection of implanted cells in the entire brain to be
visualized in high resolution." The basis of this technology is provided by genetically
altered rabies viruses. The researchers are exploiting the fact that these viruses spread
backwards via nerve cell junctions -- called synapses. The genetically altered rabies
virus, which is no longer dangerous to humans, carries a fluorescent protein.
Upon infection of the graft, the transplanted neurons turn green. At the same
time, the 'green' virus spreads backwards across established synapses to
connected host neurons, which are also turning green.
A three-dimensional nerve circuit diagram across a transparent brain
To visualize the labeled cells, the team first employed a special clearing procedure.
"This technique makes it possible to turn the brains completely transparent -- almost as
glass," says Dr. Martin Schwarz from the Bonn Department of Epileptology, who

perfected this technique. The transparent brain is then studied layer by layer, similar to
computer tomography, using what is known as a light sheet fluorescence microscope,
which Prof. Ulrich Kubitscheck and his team at the Institute for Physical and Theoretical
Chemistry at the University of Bonn developed specifically for this purpose.
"With this technique, the brain is scanned in high resolution in over 1,000 virtual optical
sections;

the

data

is

then

reconstructed

three-dimensionally,"

explains

Prof.

Kubitscheck. "As the implanted neurons and the recipient's nerve cells connected to
them light up green, a three-dimensional brain map can be created that delineates all
the recipient cells connected to the transplant -- the graft connectome," says Dr. Jonas
Doerr, who first-authored the study together with Martin Schwarz.
As the brain tissue itself becomes invisible after the clearing procedure, the researchers
in a last step aligned the fluorescent maps with neuroanatomical data generated via
magnetic resonance tomography of mouse brains. "Similar to cities on a globe, all of the
cells marked in green can thus be allocated to distinct anatomical territories," says Prof.
Mathias Hoehn from the Max Planck Institute for Metabolism Research in Cologne,
whose group conducted these calculations.
Great potential for the development of nerve cell transplants
"Our findings show that the transplanted neurons integrate in a remarkably regionspecific manner into the different transplant sites," reports Prof. Brstle. The
researchers hope that the new approach will be particularly useful for studying and
optimizing the ability of neuronal transplants to connect with the host brain before they
are used for clinical therapy. As a next step, they plan to use the rabies system to
investigate how human dopamine-producing cells can be best wired into the brain of
mice with induced Parkinson-like symptoms.

Story Source:
Materials provided by University of Bonn. Note: Content may be edited for style and
length.

Journal Reference:
1.

Jonas Doerr, Martin Karl Schwarz, Dirk Wiedermann, Anke Leinhaas, Alina
Jakobs, Florian Schloen, Inna Schwarz, Michael Diedenhofen, Nils Christian Braun,
Philipp Koch, Daniel A. Peterson, Ulrich Kubitscheck, Mathias Hoehn, Oliver
Brstle. Whole-brain 3D mapping of human neural transplant innervation. Nature
Communications, 2017; 8: 14162 DOI: 10.1038/ncomms14162

Mencias, Michelle Grace D. BSCHE 5

Biochem Engg Assignment # 1 :

31 Jan 2017
11:30-12:30 TThS

Scientists use stem cells to create human/pig chimera embryos

Date:

January 26, 2017

Source:

Cell Press

Summary:

Efforts by researchers to grow the first embryos containing cells from

humans and pigs proved more challenging than anticipated, they report in a new study.
Human/animal chimeras can offer insights into early human development and disease
onset and provide a realistic drug-testing platform. They may also someday provide a
means of growing human cells, tissues, and organs for regenerative medicine. For now,
however, they are helping scientists understand how human stem cells grow and
specialize.

This photograph shows injection of human iPS cells into a pig blastocyst. A laser beam (green
circle with a red cross inside) was used to perforate an opening to the outer membrane (Zona

Pellucida) of the pig blastocyst to allow easy access of an injection needle delivering human iPS
cells.Credit: Courtesy of Juan Carlos Izpisua Belmonte

Efforts by Salk Institute researchers to grow the first embryos containing cells from
humans and pigs proved more challenging than anticipated, they report January 26
in Cell. Human/animal chimeras can offer insights into early human development
and disease onset and provide a realistic drug-testing platform. They may also
someday provide a means of growing human cells, tissues, and organs for
regenerative medicine. For now, however, they are helping scientists understand how
human stem cells grow and specialize.
"The ultimate goal is to grow functional and transplantable tissue or organs, but we are
far away from that," says lead investigator Juan Carlos Izpisua Belmonte, a professor in
the Salk Institute of Biological Studies' Gene Expression Laboratory. "This is an
important first step."
Despite decades of work, scientists are still struggling to coax stem cells growing in Petri
dishes to become fully functional specialized adult cells, let alone three-dimensional
tissues and organs. "It's like when you try to duplicate a key. The duplicate looks almost
identical, but when you get home, it doesn't open the door. There is something we are
not doing right," says Izpisua Belmonte. "We thought growing human cells in an animal
would be much more fruitful. We still have many things to learn about the early
development of cells."
As a first step, Izpisua Belmonte and Salk Institute staff scientist Jun Wu created a
rat/mouse chimera by introducing rat cells into mouse embryos and letting them mature.
Other researchers had already created a rat/mouse chimera in 2010. That chimera was
a mouse with pancreatic tissue formed from rat cells.
Izpisua Belmonte and Wu built on that experiment by using genome editing to flexibly
direct the rat cells to grow in specific developmental niches in the mouse. To accomplish
this, they used CRISPR genome editing tools to delete critical genes in fertilized mouse
egg cells. For instance, in a given cell, they would delete a single gene critical for the

development of an organ, such as the heart, pancreas, or eye. Then, they introduced rat
stem cells into the embryos to see if they would fill the open niche. "The rat cells have a
functional copy of the missing mouse gene, so they can outcompete mouse cells in
occupying the emptied developmental organ niches," says Wu. As the organism
matured, the rat cells filled in where mouse cells could not, forming the functional
tissues of the organism's heart, eye, or pancreas.
Rat cells also grew to form a gall bladder in the mouse, even though rats stopped
developing this organ themselves over the 18 million years since rats and mice
separated evolutionarily. "This suggests that the reason a rat does not generate a gall
bladder is not because it cannot, but because the potential has been hidden by a ratspecific developmental program," says Wu. "The microenvironment has evolved through
millions of years to choose a program that defines a rat."
The team's next step was to introduce humans' cells into an organism. They decided to
use cow and pig embryos as hosts because the size of these animals' organs more
closely resembles humans than mice. The team encountered many logistical
challenges, but the scientific challenge was determining what kind of human stem cell
could survive in a cow or pig embryo.
Experiments with cow embryos were more difficult and costly than pigs, so the team
zoomed in on pigs. The effort required to complete studies of 1,500 pig embryos
involved the contributions of over 40 people, including pig farmers, over a four-year
period. "We underestimated the effort involved," says Izpisua Belmonte. "This required a
tour de force."
Not only are pigs and humans about five times more distant evolutionarily than mice and
rats, but pigs also have a gestation period that is about one-third as long as humans, so
the researchers needed to introduce human cells with perfect timing to match the
developmental stage of the pig. "It's as if the human cells were entering a freeway going
faster than the normal freeway," says Izpisua Belmonte. "If you have different speeds,
you will have accidents."

The researchers injected several different forms of human stem cells into pig embryos to
see which would survive best. The cells that survived longest and showed the most
potential to continue to develop were "intermediate" human pluripotent stem cells. Socalled "nave" cells resemble cells from an earlier developmental origin with unrestricted
developmental potential; "primed" cells have developed further, but still remain
pluripotent. "Intermediate cells are somewhere in between," says Wu.
The human cells survived and formed a human/pig chimera embryo. Embryos were
implanted in sows and allowed to develop for between three and four weeks. "This is
long enough for us to try to understand how the human and pig cells mix together early
on without raising ethical concerns about mature chimeric animals," says Izpisua
Belmonte.
Even using the most well-performing human stem cells, the level of contribution to the
chimerized embryo was not high. "It's low," says Wu.
Izpisua Belmonte considers this good news. One concern with the creation of
human/animal chimeras is that the chimera will be too human. For instance, researchers
don't want human cells to contribute to the formation of the brain.
In this study, the human cells did not become precursors of brain cells that can grow into
the central nervous system. Rather, they were developing into muscle cells and
precursors of other organs. "At this point, we wanted to know whether human cells can
contribute at all to address the 'yes or no' question," he says. "Now that we know the
answer is yes, our next challenge is to improve efficiency and guide the human cells into
forming a particular organ in pigs."
To do this, the researchers are using CRISPR to perform genome editing on the pig
genome, as they did with mice, to open gaps that human cells can fill in. The work is in
progress.

This research study was supported by The Fundacin Sneca in Murcia, Spain, the
Universidad Catlica San Antonio de Murcia (UCAM), the Fundacion Dr. Pedro Guillen,
the G. Harold and Leila Y. Mathers Charitable Foundation, and The Moxie Foundation.

Story Source:
Materials provided by Cell Press.
https://www.sciencedaily.com/releases/2017/01/170126132536.htm
(Retrieved: 29 Jan 2017)

Journal Reference:
Jun Wu, Aida Platero-Luengo, Masahiro Sakurai, Atsushi Sugawara, Maria

1.

Antonia Gil, Takayoshi Yamauchi, Keiichiro Suzuki, Yanina Soledad Bogliotti, Cristina
Cuello, Mariana Morales Valencia, Daiji Okumura, Jingping Luo, Marcela Vilario,
Inmaculada Parrilla, Delia Alba Soto, Cristina A. Martinez, Tomoaki Hishida, Sonia
Snchez-Bautista, M. Llanos Martinez-Martinez, Huili Wang, Alicia Nohalez, Emi
Aizawa, Paloma Martinez-Redondo, Alejandro Ocampo, Pradeep Reddy, Jordi Roca,
Elizabeth A. Maga, Concepcion Rodriguez Esteban, W. Travis Berggren, Estrella Nuez
Delicado, Jeronimo Lajara, Isabel Guillen, Pedro Guillen, Josep M. Campistol, Emilio A.
Martinez, Pablo Juan Ross, Juan Carlos Izpisua Belmonte. Interspecies Chimerism
with

Mammalian

Pluripotent

DOI: 10.1016/j.cell.2016.12.036

Stem

Cells. Cell,

2017;

168

(3):

473