They're messin with your brain! Ha, Ha! Not quite yet.

From: Michael Ragland (ragland666_at_webtv.net)
Date: 10/30/04

Next message: Brett Aubrey: "Re: Time without end: Physics and Biology in an Open Universe"
Previous message: Guy Hoelzer: "Re: the why question"
Messages sorted by: [ date ] [ thread ]

Date: Sat, 30 Oct 2004 17:32:01 +0000 (UTC)

doi:10.1038/nn1101-1156
November 2001 Volume 4 Number Supp pp 1156 - 1158

An impulse to the brain—using in vivo electroporation

Takayoshi Inoue & Robb Krumlauf

The authors are in the Stowers Institute for Medical Research, 1000 East
50th Street, Kansas City, Missouri 64110, USA.
e-mail: rek@stowers-institute.org

To understand the mechanisms and processes that underlie neural
development, plasticity, physiology and function, it is essential to be
able to monitor and manipulate the behavior of cells over time and to
modify gene expression in viable cells, embryos and tissues. Recent
progress in a number of technologies, including in vivo imaging (see
article in this issue by Lichtman and Fraser), embryo culture and gene
transfer by in vivo electroporation (EP), are now permitting us to
approach important questions in a new way. Here we summarize some of the
potential uses and advantages of combining in vivo EP with other methods
for neuroscience research.

Efficient gene transfer by the EP technique has been widely used to
introduce exogenous molecules into both prokaryotic and eukaryotic
cells. Although the detailed mechanisms are unknown, transient pores
generated by electric shocks at the cellular membrane allow charged
macromolecules such as proteins, RNA and DNA to actively penetrate into
cells by means of electrophoresis. The difficulty in applying this
approach to living tissues or organisms had been that the electric
pulses often damage cells and result in substantial cell death. A key
breakthrough was the discovery that a rapid series of controlled square
wave pulses, instead of averaged or bell-shaped exponential pulses,
dramatically reduced the levels of cell death. In 1997, Muramatsu and
colleagues first reported remarkable results using in vivo
electroporation in developing chicken embryos1. This important and
convenient technology is now routinely being used by chick embryologists
and is also being applied to many other living tissues and organisms,
including mammals2-6. Although much of the current use centers on early
development, this method is equally applicable to adult tissues, organs
or differentiated post-mitotic cell populations.

How does this new technology bring benefits to the field of
neuroscience? In vivo EP has several advantages for investigating neural
development, as it facilitates and complements both genetic and
manipulative approaches in many experimental systems. The neural tube or
brain vesicles are particularly easy to target, as a gene expression
vector can be placed in the lumen, permitting the directed transfer of
DNA, which carries a negative charge, to the side of the positive
electrode (Fig. 1a)2-4, 6. If an enhancer/ promoter combination (for
example, rous or cytomegalo virus) capable of directing expression in
most cell types is used, it is routinely possible to obtain expression
in 10–100% of transfected cells by optimizing conditions through
variation of voltage and numbers of pulses (Fig. 1a). It is also
possible to target specific cell types or restricted populations through
the use of enhancers capable of mediating spatial, temporal or
tissue-specific expression2. Furthermore, the EP technique provides an
effective approach for mapping and identifying cis-regulatory elements2,
7. However, there can be variability in expression with some
electroporated regulatory regions, presumably because the electroporated
DNA generally does not become integrated. This may be analogous to the
differences seen in experiments using transient transfection versus
stable cells lines or transgenes.

A further refinement of the EP approach arises from variations in types
of electrodes and application of the voltage. Specific promoter/enhancer
elements are not the only way to modulate gene expression within
restricted tissues and/or groups of cells, as the targeting can also be
directed by careful positioning of the electrodes to apply the electric
field in a specific manner. By using electrodes of different size or
type (from a fine point to a long wire or plate) or modifying the
voltage, it is possible to control the relative size of the transfected
area, which can vary from a single cell to entire tissues4, 5, 8, 9. If
the DNA can be injected in the proper position, the electrodes do not
always need to be placed in the embryo itself, as long as a current is
passed through the relevant tissues (Fig. 1a and b). This permits a wide
spectrum of electrode orientations to enable gene transfer along
different axes (anteroposterior, dorsoventral, mediolateral, left-right,
proximodistal). By performing the operation at different or multiple
stages in the embryo or adult tissues, one can control the timing of
expression for exogenous genes in vivo to study many aspects of neural
differentiation and patterning9, 10. Multiple genes can simultaneously
be examined by co-electroporation of mixed expression vectors2, 11 and
blocking gene function or expression is also possible using dominant
negative molecules12. The expression of fluorescent reporter genes can
act as lineage tracers in grafting and fate-mapping experiments6.
Furthermore, different combinations of these tools or approaches can be
used with multiple rounds of electroporation on the same embryo, as
viability or integrity of tissues and embryos is high using the EP
technique. These analyses can be done in wild-type or mutant embryos,
which will enhance approaches to studying normal and mutant cell
behaviors and phenotypes. Hence, combined with classical manipulative
advantages in chicken or powerful genetics in mouse, EP will allow the
development of new approaches to the analysis of gene functions and
regulatory mechanisms in the nervous system.

Recently, in mammals, EP has been combined with whole-embryo culture,
which permits the manipulated embryo to develop normally for a few days
in vitro4. Using this combination, it is possible to target expression
of a green fluorescent protein (GFP) reporter to restricted territories
in the developing central nervous system (CNS) of mouse embryos
harvested at different stages (Fig. 1b). Taking advantage of this
method, the mechanisms of early brain compartmentalization11,
specification of the neuronal fate13 and cellular migration in CNS14
have already been studied. A disadvantage of whole embryo culture in the
mouse is that the method is limited to events between E7.0–13.0, as it
is difficult for the placenta to develop efficiently in vitro. However,
an exciting possibility for the future is that methods for in utero15 or
ex utero16 development exist that could equally be applicable to EP and
manipulation of embryos, as it is possible to properly target the
electrodes. In this regard, the ultrasound backscatter microscope is
effective at visualizing developing embryos17 and should help in
perfecting the transfer genes in utero through electroporation. This
technique can also be extended to slice cultures of adult brains or
organ culture to investigate questions dealing with events after the
mammalian CNS is formed.

doi:10.1038/nn1101-1158
November 2001 Volume 4 Number Supp pp 1158 - 1159

Single neuron labeling and genetic manipulation

Liqun Luo & Hui Zong

The authors are in the Department of Biological Sciences, Stanford
University, Stanford, California 94305, USA.
e-mail: lluo@stanford.edu

Those who have observed brain sections stained by the Golgi method would
agree with Ramon y Cajal1: "What an unexpected sight!...everything is
simple, clear and unconfused. There is no longer any question of
interpretation." The Golgi method labels a very small population of
random neurons in their entirety in an otherwise unstained brain,
allowing visualization of dendritic trees of individual neurons and
tracing of long distance axonal projections1. It is difficult to
overestimate the enormous contribution this method has brought to
neuroscience.

Now imagine that one can use genetic manipulation to create, at will,
singly-labeled neurons in intact brain tissue or in vivo, and moreover,
knock out endogenous genes in only these labeled neurons. This will help
us to assess the functions of genes in single clearly labeled neurons,
increasing the power of phenotypic detection; avoid pleiotropic effects
of genes by focusing on the tissue and developmental stages of interest;
and determine cell-autonomy of gene action. The cellular and molecular
mechanisms that ensure the elaborate connection and function of the
nervous system can then be dissected with single neuron resolution.

How can one achieve this purpose? Genetically mosaic animals, in which a
subset of somatic tissues have different genotypes compared to the rest
of the organisms, have long been used to attack biological problems in
Caenorhabditis elegans, Drosophila and mice. Traditionally, genetic
mosaic animals were generated via spontaneous or X-ray-induced mitotic
recombination, resulting in progeny homozygous mutant for a candidate
gene of interest in the heterozygous (and therefore phenotypically
normal in most cases) background. In mice, chimaeras can also be
generated by mixing embryonic cells of different genotypes. With the
introduction of sequence-specific recombination systems such as FLP/FRT
or Cre/LoxP, not only can one dramatically increase the efficiency of
generating mosaic animals, but also control where and when such
recombination occurs by dictating the expression pattern of the FLP or
Cre recombinase.

Mosaic analysis is only useful if one can tell apart mutant cells from
wild-type cells. In Drosophila, for instance, traditional mosaic
analysis relies on external markers such as body color or bristle shape.
To make it versatile, a cell marking system was introduced in the highly
efficient FLP/FRT system2 by placing a marker gene on the chromosome arm
in trans to the mutation of interest, both distal to the homologous FRT
sites. After mitotic recombination, homozygous mutant cells become the
only cells in mosaic animals that do not express the gene, and hence are
uniquely unlabeled3. FRT transgenes have been inserted at the bases of
all chromosome arms to allow mosaic analysis of vast majority of genes3.
Although very useful in studying many developmental biology problems, it
is not ideal to study complex neuronal morphogenesis, as one cannot
visualize mutant neurons.

The MARCM system4 (for mosaic analysis with a repressible cell marker)
in Drosophila solved this problem. Taking advantage of the highly
successful GAL4/UAS binary expression system in Drosophila5, we
introduced the GAL80 protein, an inhibitor of GAL4, into flies under the
control of a ubiquitous promoter. The GAL80 transgene was placed on the
chromosome arm in trans to the mutation of interest. The generation of a
homozygous mutant cell is therefore coupled with the loss of the GAL80
transgene, and hence allows the marker gene expression. In this way,
only homozygous mutant cells are uniquely labeled in the mosaic animal4.
By controlling where and when the FLP recombinase is expressed, uniquely
labeled single mutant neurons can be generated routinely. The MARCM
system has been used to study cell lineage, analyze gene function in
axon and dendrite development, isolate new mutants affecting complex
neural developmental processes, and study neural network assembly6-8.

The use of MARCM is not limited to studying neural development. For
instance, a GAL4/UAS-based system has been described to conditionally
inactivate synaptic transmission—by a simple temperature shift
resulting in the expression of dominant temperature-sensitive mutant
protein involved in synaptic vesicle endocytosis—in all neurons that
express GAL49. This has proven very useful to identify the roles of
specific groups of neurons in specific behaviors10-12. It is conceivable
that one can further map the function of individual neurons important
for a specific behavior by combining this technique with the MARCM
system.

How about mice? Through homologous recombination, endogenous genes can
be removed in otherwise wild-type mice13. However, powerful, standard
gene targeting has limitations: many genes have pleiotropic functions
that impede the analysis of their nervous system functions. `Conditional
knockout´ based on Cre/loxP site-directed homologous recombination has
been used be to remove genes selectively in particular developmental
stages or a subset of tissues14, 15. FLP/FRT has also been used16. As
discussed above, it will be very useful if one can delete genes in
isolated neurons and simultaneously visualize these mutant neurons.
Using the current technology15, one can, in theory, uniquely label
homozygous null mutant cells in the Cre/Lox system by inserting a marker
gene after the transcription stop in the `floxed´ allele. Most
Cre-mediated recombination is `too efficient´ to generate
single-neuron mosaics. However, Cre-expressing mice generating very low
efficiency recombination have been reported, capable of labeling
isolated single neurons in vivo (personal communication, J. Huang, Cold
Spring Harbor and S. Tonegawa, MIT). Still, there are several technical
limitations in applying the above method other than the intense labor
needed to generate these mice individually. Could a systematic way of
generating mosaic animals be established by making use of mitotic
recombination between homologous chromosomes in mice, as in Drosophila?
The biggest unknown seems to be whether one can achieve a sufficiently
high recombination rate between homologous chromosomes in somatic cells
of mice.

"Tiny green men might have been a better experiment."

Stephen Hawking
(paraphrasing from a "Universe in a Nutshell".

Next message: Brett Aubrey: "Re: Time without end: Physics and Biology in an Open Universe"
Previous message: Guy Hoelzer: "Re: the why question"
Messages sorted by: [ date ] [ thread ]

Relevant Pages

Designer protein tackles HIV
... Designer protein tackles HIV ... Enzyme could create tailor-made cells to be injected back into ... By creating a custom-designed enzyme that can sever a gene, ...
(soc.culture.zimbabwe)
Re: Genetically engineered micro-organisms to fight disease?
... >works by changing patients own gene to do some beneficial work. ... It works by extracting cells from a patient who lacks functional copies ... >phage whatever) that homes in on cancer cells or whatever and destroys it. ... There has been talk of developing bacteriophages to attack pathogenic ...
(sci.med)
Re: Genetically engineered micro-organisms to fight disease?
... >works by changing patients own gene to do some beneficial work. ... It works by extracting cells from a patient who lacks functional copies ... >phage whatever) that homes in on cancer cells or whatever and destroys it. ... There has been talk of developing bacteriophages to attack pathogenic ...
(sci.med.diseases.cancer)
Re: Gene expression questions
... I was just curious if cells make assumptions as to how to translate & ... A brief answer to your question is: transcription factors. ... gene so that it can be expressed. ...
(sci.bio.evolution)
Answers to quiz + ASAT capability & the future of space itself
... What are dominant and recessive characteristics? ... characteristic requires only one gene to be present whereas a both ... What are T cells? ... ABM systems are also ASAT systems and the most popular is a laser ...
(sci.space.policy)