Transgenesis Techniques. Principles and Protocols

436 Pages·1993·24.584 MB·English

by  David Murphy, David A. Carter

CHAPTER1
Introduction to Thnsgenesis
David Murphy and David A. Carter
Over the past decade, a number of techniques have been developed
that allow the introduction of defined, cloned DNA sequences into
animal germ lines. Once inserted, these sequences, now called trans-
genes, are stably passed on from generation to generation. In other
words, the transgene becomes a part of the genetic make-up of that
particular line of animal. Every individual of a particular line will
carry the transgene in every cell of its body. Of fundamental impor-
tance has been the observation that transgenes are often expressed-
that is they are functional-and that this expression is subject to correct
tissue-specific, developmental, and physiological regulation. It is
therefore now possible to analyze the role and regulation of specific
cloned genes within the whole organism. Such organisms are called
transgenic organisms.
Of all technical achievements that have advancedb iological sciences,
few have opened up such possibilities as transgenic methodologies.
The ability to change selectively the genetic make-up of a multicellular
organism and thereby permanently alter the activity of particular pro-
teins has important bearing on all areas of biological investigation. The
extension of transgenic techniques to mammalian speciesh as caught the
imagination of both the screntific and general communities, providing
From Methods tn Molecular Biology, Vol 18 Transgenests Techntques Prmclples and Protocols
Edtted by D Murphy and D A Carter Copynght 01993 Humana Press Inc , Totowa, NJ
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4 Murphy and Carter
causef or optimism and, at the same time, some concern when the impli-
cations for medicine and agriculture are considered.
Despite the notoriety of transgenic animals, the techniques for pro-
ducing transgenics are not widely available in research laboratories,
being found associated with the developmental biology laboratories
where the techniques originated. The primary aim of the present vol-
ume is to facilitate the expansion of transgenic experimentation such
that it becomes a commonly available approach. To this end, all of
the techniques required for both the production and analysis of trans-
genie animals are described. Although a number of basic molecular
cloning techniques are described herein, additional reference may be
required, however, for some techniques used m the building of
transgene constructs. The reader is referred to other volumes m this
series (e.g., ref. 1).
The emphasis of the present volume is on transgenic mice, since
this species has been the principal model in most transgenic studies.
Comprehensive details for the production of transgenics in two other
mammalian species, namely rats and sheep, are also included. Rats
are included since they are the model of choice in many physiologi-
cal and pharmacological studies, and sheep are included as an exam-
ple of the genetic manipulation of livestock. In addition, techniques
and applications of transgenesis in two nonmammalian species are
also presented.
The superiority of a transgenic approach compared with the use of
models such as cultured cells, for example, is clear to anyone inves-
tigating complex biological systems. However, the most appropriate
design of transgene experiments is, in our experience, not always so
clear to investigators with no experience of this approach. Thus, the
results of a lengthy (the time-scale of transgenic experiments is in
months and years rather than days) first-time study may simply inform
the investigator that he or she should have spent more time thinkmg
about the construction of the transgene or that a different strategy
would have better answered the question. We feel that a knowledge
of the transgenic literature is essential to the successful adoption of
this technology into a novice laboratory. As an introduction to the
field, we have therefore included short review chapters that consider
the application of transgenesis to three selected complex systems,
namely the brain, the immune system, and cancer. An additional intro-
Introduction to Transgenesis 5
ductory chapter considers the commercial applications of transgenesis,
with particular emphasis on the genetic manipulation of sheep. It is
hoped that with this additional insight, new investigators will be bet-
ter equipped to use the techniques described here, and to apply trans-
genesis to its maximum potential.
Legal Obligations of Researchers
and Animal Welfare
Many of the procedures described in the present volume are the
subject of governmental regulation. Investigators should consult the
appropriate local authorities before embarking on any study involv-
ing animal experimentation and/or genetic engineering. Researchers
must always consider the fundamental principle of ethical research
(2), which is that experimental animals must not be subjected to avoid-
able distress or discomfort. Research animals should be acquired and
cared for in accordance with standards established by the National
Institutes of Health (3).
References
1. Walker, J. M. (ed.) (1984) Methods in Molecular Biology, vol. 2: Nucleic Acids.
Humana, Clifton, NJ.
2 Handbookfor the Use ofAnimals in Neuroscience Research. (1991) Avalable
from the Society for Neuroscience, Publication Orders, 11 DuPont Crrcle, N.W.,
Suite5 00, Washington, DC 20036.
3. NIH Guide for the Care and Use ofLaboratory Animals, rev. ed. (1985) National
Institutes of Health Publication No. 85-23, Washmgton, DC.

CHAPTER2
Transgenic Rodents
and the Study
of the Central Nervous System
David A. Carter
1. Introduction
The following view of transgenic studies applied to the understand-
ing of brain function is written as a guide for neuroscientists who
may be considering transgenesis techniques in the pursuit of their
research. Although most important studies are covered, the chapter is
not a comprehensive review of the literature. Rather, it is intended to
convey the possibilities of a particular technique, and thus provide an
indication of both strategies and attainable goals. In addition to pro-
viding information relevant to experimental design, particular areas
of neuroscience that have benefited from transgenic approaches will
also be discussed. Developmental neurobiology is not specifically
addressed; for a recent review of genomic manipulations in neuron/
glial lineage analysis, see ref. 1. The first section of the present chap-
ter describes the use of transgenics to localize cis-acting elements
within neuronal genes, which act in the mediation of cell-specific
and regulated expression, Transgenic mice have been chosen, to an
extent, by default as models for neuronal gene analysis since suit-
able, permanent neuronal cell lines are not available for transfection
studies. Analysis of enhancer/promoter regions is a daunting under-
taking in transgenics and there is a strong argument for combining
these studies with DNA-mediated transfection experiments in heter-
From Methods m Molecular Wology, Vol 18 Tramgenesis Technrques Prmples and Protocols
Edlted by D Murphy and D A. Carter Copynght 01993 Humana Press Inc , Totowa, NJ
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Carter
ologous cell lines. Transgenic animals are, however, much more than
model expression systems; they provide a unique opportunity to study
both the regulation and role of neuronal genes in the context of inte-
grated brain systems. The second section deals with approaches to
neuronal gene function, concerning both gain-of-function and loss-
of-function techniques. In the third section, the use of transgenics in
the molecular analysis of neurological syndromes is discussed: Are
transgenic rodents useful models for human brain diseases? Finally,
the capacity to generate neuronal cell lines following targeted expres-
sion of oncogenes to specific neurons is assessed.T he transgenic stud-
ies discussed here employ mice as the experimental animal, and, with
one exception, use DNA microinjection techniques (see Chapters 18
and 19) in the generation of transgenics. At the time of writing, no
studies have been published in which neuronal transgenes are
expressed in rats. However, the techniques for producing transgenic
rats are now available (see Chapters 26-3 1); given the ubiquitous use
of rats by neuroscience investigators it is anticipated that the rat will
be increasingly used to provide transgenic models for brain research.
2. Characterization of Regulatory
Regions/Elements in Neuronal Genes
The primary aim, indeed the sine qua non, of transgenic studies is to
direct expression of particular genes to specific groups of cells within
heterogeneousc ellular systems. Cell/tissue-specific expression is both
an essential prerequisite to functional analysis of transgene expres-
sion and a requirement of studies in which the role of c&acting regu-
latory elements are investigated in an appropriate cellular context;
i.e., in the presence of corresponding trans-acting factors. The multi-
plicity of cell-types in the mammalian brain, each exhibiting unique
patterns of gene expression, represents the extreme example in biol-
ogy of a complex system in which precise genetic control is essential
to permit the variety of phenotype. Although cell-specific transgene
expression has been attained in a number of studies on the mouse
central nervous system (CNS), it is not possible to make specific rec-
ommendations on the design of recombinant gene constructs for use
in transgenic studies on the brain. On the contrary, comparison of
different experiments has revealed considerable variation in the size
and make-up of DNA constructs that appear to be required to direct
Transgenic Rodents and the CNS 9
neuronal expression of particular genes. However, the literature is
now of sufficient breadth to allow some useful generalizations to be
drawn and questions may reasonably be posed regarding the size and
composition of DNA constructs.
3. Design of DNA Constructs
for Neuronal Transgene Expression
The quick, and probably most appropriate, answer to the question
“How big a piece of DNA must be used to give neuron specific expres-
sion?’ is “As big as possible”; in other words, use the largest avail-
able clone. Unlike other techniques, in which genetic information
may be transmitted to experimental animals by viruses, for example,
the microinjection technique offers no physical limit on the size of
DNA construct used. Our current understanding of transcriptional
control mechanisms, which suggests the presence of regulatory ele-
ments throughout the transcription unit, including elements >lO kb 5’
to the transcriptional start site, certainly supports the “more is better”
approach, Since neuron-specific regulatory elements have not been
identified and, of course, complete sequence of the investigated gene
may not be available, such an approach appears justified. It is also
supported by experimental findings; for example studies on the SCGlO
gene (2; see also Table 1) have revealed that while constructs con-
taining either 3.5 or 4.2 kb of 5’ flanking sequence direct neuron-
specific expression of a reporter gene, use of only 0.55 kb of proximal
promoter sequence results in a “relaxed” or deregulated pattern of
expression, which includes nonneuronal tissues. Since reporter levels
still remained highest in the brain, it is apparent that neuron-specific
expression of SCGlO may be achieved through selective repression
in other tissues mediated by silencer elements in distal 5’ regions (2).
The results of another study (3; see also Table 1) are consistent with
a similar interpretation with respect to specific patterns of peripheral
transgene expression. The latter study and others (e.g., 4, see also
Table 1) are, furthermore, consistent with distal 5’ elements regulat-
ing neuronal expression since, in both cases, appropriate expression
is found in peripheral tissues but not in the brain. Although more
studies are required, some general features do appear to be emerging,
namely that neuronal expression is specified by regulatory sequences
additional to those that permit nonneuronal expression. The location
10 Carter
Table 1
Expression of Neuronal Transgenes m Mice
Flanking DNA Neuronal expn. Peripheral expn.
Gene 5’ 3’ +/- Sp. EC. +/- sp. EC. Ref
SCGlO/CAT (r) 4.2 0 + ND ND t t - 2
3.5 0 + ND ND t t 2
0.55 0 t ND ND + t t 2
NSEllacZ (r) 1.8 0 tt - - += 5
NGF (h) 8.0 7.5 t+ - ;D ND ND 7
L7/lacZ (m) 4.0 2.0 t t -1PN - - - 8
GnRH (m) 5.0 3.5 t t -PVN t t t 9
VP/Tag (b) 1.25 0 -- - t - t 18
VP/CAT (b) 1.25 0 t- + t - t 20
OT t- t t - t 12
OT/VP ((br)) 00..63 6/1.4 i*” tt - - - - 11
PNMT (h) 2.0 4.0 -- - t t t 3
PNMT (h) 8.0 4.0 -- - t t 3
POMClneo (r) 0.77 0 -- - t t - 4
GRF/Tag (h) 1.6 0 -- - + - t 19
GRF/NGF (h) 1.6 0 t ND ND - - - 19
GRFkHras (h) 1.6 0 t ND ND - - - 19
DBH/lacZ (h) 5 8 0 + + tb + t + 6
Abbreviations CAT, chloramphemcol acetyltransferase, DBH, dopamme P-hydroxylase,
EC , ectopic expression; GnRH, gonadotrophin hormone-releasing hormone, GRF, growth
hormone releasing factor, IPN, mterpeduncular nucleus only, NSE, neuron-spectftc eno-
lase, PNMT, phenylethanolamme N-methyltransferase, PVN, paraventrrcular nucleus only;
Sp., cell-specific expresston, Tag, SV 40 large-T anttgen
ND Not determined/reported
Promoters are (b) bovme, (h) human, (m) mouse, (r) rat
aTestrcular expression (often observed as ectoprc site of expresston m transgemcs with
neuronal promoters)
bSee ref. 6 for excellent drscussron of mechamsms of ectoprc expression
of such sequences may, however, be highly variable; whereas 8 kb of
5’ flanking sequence was not sufficient to direct expression of the
phenylethanolamine N-methyl-transferase gene (PNMT) to neurons
in the brainstem (3), a construct containing only 1.8 kb of the neu-
ron-specific enolase gene (NSE) resulted m panneuronal expression
of a P-galactosidase marker, with peripheral expression in testis only
(5). Furthermore, with respect to the PNMT transgene (3), a recent
study (6) has shown that a construct containing only 5.8 kb of 5’
sequence from the closely related dopamine P-hydroxylase (DBH)
Transgenic Rodents and the CNS 11
gene was sufficient to direct specific expression to noradrenergic
neurons in the brainstem. A more sophisticated approach to design-
ing constructs for neuronal transgenesis studies would include a con-
sideration of factors other than simply the length of 5’ flanking
sequence. First, the possible presence of regulatory elements within
intronic, exonic, and 3’ flanking regions should be borne in mind. It
is noteworthy that some of the successful transgenic studies employ-
ing neuronal genes (7-9; see also Table l), have used constructs that
include all of these regions. For example, 8 kb of the mouse L7 gene
was sufficient to direct a highly specific, endogenous pattern of
transgene expression to the mouse brain with a low level of ectopic
expression in a single brain nucleus (8). Second, regulatory elements
may be located in adjacent, linked genes. This phenomenon, which
has been described for globm genes (lo), is apparent for the neuroen-
docrine peptide genes vasopressin (VP) and oxytocin (OT). Construc-
tion of a “mini-locus” transgene containing 5’ flanking sequence of
both VP (1.4 kb) and OT (0.36 kb) resulted in cell-specific expres-
sion of OT in the mouse hypothalamus; VP was not detected (II).
Previously, an OT construct containing 0.6 kb of flanking sequence
failed to produce an appropriate expression pattern (22; see also Table
l), indicating that correct expression of OT is dependent on sequences
contained in the VP gene. Further studies are required to gain an un-
derstanding of the full extent of regulatory interactions between these
linked genes. Recent transgenic studies have therefore described pro-
moters that may be used to direct transgene expression in a brain-
specific manner. Aside from the practical applications in potential
functional investigations, these studies have provided a basis for under-
standing the mechanisms that underlie neuron-specific gene expres-
sion. Following the localization of regulatory elements to relatively
large regions of DNA, it is anticipated that subsequent studies will
lead to the precise delineation of these elements. For example, the pan-
neuronal expression pattern obtained using the NSE gene (5) and the
Thy- 1 gene (13) may allow the identification of neuron-specific ele-
ments and truns-acting nuclear proteins. At the presentt ime neuron-spe-
cific control elements have not been described, although some interest
has been focused on the octameric sequenceS -GCCCAGCC-3’, which
is present in the proximal promoter region of several neuronally
expressed peptide genes (24,15). Characterization of such elements
12 Carter
may provide powerful tools for future studies. Currently, promoters
such as NSE and Thy-l may be used to transform neurons in a non-
specific manner. The L7 promoter (8), on the other hand, is an example
of a promoter that may be used to manipulate selectively the pheno-
type of specific groups of brain cells.
4. Ectopic Neuronal Expression
of Chime& Transgenes
Ectopic expression of transgenes in cells that do not express the
corresponding endogenous gene has been found in a number of trans-
genie studies in which chimeric “fusion” gene constructs have been
employed. Analysis of this phenomenon has revealed that the unex-
pected pattern of expression results from a combinatorial action of
&-acting elements from both promoter and reporter regions of the
transgene, which cannot be predicted from the expression patterns of
the individual elements (16). For example, a construct containing the
mouse metallothionein-I (MT-I) promoter linked to either the rat or
human growth hormone gene produced a unique pattern of expres-
sion in several brain regions (17). This surprising result is clearly of
interest with regard to the developmental relationships between dif-
ferent groups of neurons and may provide novel insights into devel-
opmental events. However, ectopic expression is not desirable m
studies which are designed to target specific groups of neurons. First,
the expression pattern obtained may be entirely inappropriate. Thus,
transgenics produced with neuronal promoters linked to SV 40 large
T-antigen sequence exhibited no transgene expression in brain, rather
tumors and hyperplasia in ectopic peripheral sites (l&19; see also
Table 1). Analysis of alternative constructs, in which different “report-
ers” were used, resulted in neuronal expression (19,20; see also Table
1) indicating that the ectopic pattern obtained with the initial con-
structs may result from a unique synergistic interaction between the
two parts of the chimeric gene. Second, even in casesw here the desired
brain expression is obtained, the severe consequences of simultaneous
ectopic expression may confound interpretation of the experiment as
a result of either secondary effects or impaired health of the animal.
It may be possible to prolong the experiment; for example, thymec-
tomy of mice exhibiting thymic hyperplasia (19) increased the sur-
vival time of transgenics expressing a growth hormone releasing factor