Brain Research Protocols 5 Ž2000.

159–166
www.elsevier.comrlocaterbres

Protocol

Expression analysis of Escherichia coli lacZ reporter gene in transgenic mice

Eiki Takahashi a,b,) , Norimasa Miyamoto a , Noriko Kajiwara b , Keiko Furuya b ,
Keiko Yanai-Taniguchi b , Fumihiro Sugiyama b , Ken-ichi Yagami b
a
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Tsukuba, Ibaraki 300-2635, Japan
b
Laboratory Research Animal Center, UniÕersity of Tsukuba, Tsukuba, Ibaraki 305-8527, Japan
Accepted 18 January 2000

Abstract

To define a gene expression mechanism, it is often advantageous to use a reporter gene and transgenic mouse. The lacZ reporter gene
is particularly useful for studies of the cis-regulatory element for tissue-specific expression in transgenic mice because of the ease of the
enzyme assay and visualization on sections. In this report, we describe our method for examining the cis-regulatory element in transgenic
mice, including choice of the lacZ gene, generation of transgenic mice, and analysis of b-galactosidase activity. q 2000 Elsevier Science
B.V. All rights reserved.

Themes: Cellular and molecular biology

Topics: Staining, tracing, and imaging techniques

Keywords: lacZ; Transcriptional regulation; cis-Regulatory element; Transgenic mice

1. Type of research 3. Materials and methods

Ža. Analysis of cis-regulatory element.

Žb. Detection of gene expression.
2. Time required
Ža. Construction of transgene: 1–4 weeks.
Žb. Generation of transgenic mice from injection to
founder: 7–12 weeks.
Žc. Generation of transgenic mice from founder to F1:
8–12 weeks.
Žd. Analysis of mice by PCR: 1–2 days.
Že. Assay of b-galactosidase activity: 1–2 days.
Žf. Staining to detect b-galactosidase activity: 1–2 days.
)
Corresponding author. Tsukuba Research Laboratories, Eisai Co.,
Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan. Fax: q81-298-
47-2037; e-mail: e2-takahashi@hhc.eisai.co.jp
Animals. ICR mice: Charles River, Japan.
Chemicals and reagents. All reagents used were of
analytical grade and were purchased from Sigma ŽSt.
Louis, MO, USA. unless otherwise stated.
Ø Sodium pentobarbital ŽNembutal. ŽAbbott, North
Chicago, IL, USA..
Ø b-Galactosidase assay kit: kit including ortho-
nitrophenyl-b-D-galactopyranoside ŽONPG., Cleavage
buffer, b-mercaptoethanol ŽInvitrogen, Carlsbad, USA..
Ø Fixation solution: 2% paraformaldehyde and 0.5% glu-
taraldehyde in 0.1 M phosphate buffer, pH 7.4.
Ø 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside ŽX-
gal. ŽTakara Shuzo, Tokyo, Japan..
Ø Proteinase K solution ŽGibco BRL, Gaithersburg, MD,
USA..
Ø OCT compound ŽSakura Finetek, Tokyo, Japan..
Ø X-gal staining solution: 0.5 mgrml X-gal, 5 mM potas-
sium ferrocyanide, 5 mM potassium ferricyanide and 1
mM MgCl 2 in 0.1 M phosphate buffer, pH 7.4.

1385-299Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 5 - 2 9 9 X Ž 0 0 . 0 0 0 0 9 - X
 160
E. Takahashi et al.r Brain Research Protocols 5 (2000) 159–166
Fig. 1. Schematic illustration of the protocol. ŽA. Generation of transgenic mice. ŽB. Assay for b-galactosidase activity in whole tissue. ŽC. Staining to detect b-galactosidase activity on sections.
 E. Takahashi et al.r Brain Research Protocols 5 (2000) 159–166
Ø Nuclear fast red ŽVector Laboratories, Burlingame,
USA..
4. Detailed procedure
The procedure is illustrated schematically in Fig. 1A,B
and C.
4.1. Construction of transgene
To dissect the molecular mechanisms underlying the
neuron-specific expression of the mouse PrQ-type Ca2q
channel a 1A subunit gene using transgenic mice, we con-
structed the transgene a 1A 6.3-lacZ, including approxi-
mately 6.3 kb of the 5X-upstream region of the a 1A subunit
gene fused to the Escherichia coli lacZ reporter gene. The
lacZ reporter gene, including lacZ, SV40 small T splice
and polyadenylation sequences, was fused at the restriction
site present in the exon so as to maintain the translation
site of a 1A subunit gene. The transgene a 1A 6.3-lacZ was
isolated from the vector backbone by digesting the plasmid
pa 1A 6.3-lacZ with NotI and BamHI. The construction of
the plasmid pa 1A 6.3-lacZ has been reported in detail
elsewhere w19x. Unique restriction sites should be main-
tained at both ends of transgene to allow isolation from the
plasmid vector backbone before injection into fertilized
mouse eggs. In the absence of a suitable restriction site,
this can be created by PCR.
4.2. Generation of transgenic mice
Transgenic mice were produced accordingly to standard
procedures w4x ŽFig. 1A.. The transgene was removed from
the vector backbone. The transgene was purified from an
agarose gel using glass powder and filtered through 0.2-mm
filters. The concentration of injected transgene solution
was 5 mgrml Žin 10 mM Tris–HCl, 0.1 mM EDTA, pH
7.4.. The transgene was injected into the male pronuclei of
200–400 fertilized eggs. After injection, the eggs were
transferred into surrogate pseudopregnant females for de-
velopment and delivery. The transferred eggs gave rise to
20–50 offspring and represented at least two transgenic
founders.
Table 1
Expression of b-galactosidase activity in various tissues of transgenic mice
b-Galactosidase activity was expressed as unitsrminrmg protein.
Lines Copy No. Brain
Wild-type – –
a 1A 6.3-lacZ-14 2 28.8 " 5.3
a 1A 6.3-lacZ-15 5 21.0 " 0.6
a 1A 6.3-lacZ-16 2 –
a 1A 6.3-lacZ-30 2 13.5 " 3.2
161
4.3. Identification of transgenic mice
Transgenic mice produced by this procedure were iden-
tified by PCR from tail DNA biopsies. After 10-min
incubation of tail DNA at 958C in 100 ml of distilled
water, 1 ml of proteinase K solution was added. After a
2-h incubation at 558C, 5 ml of the heated-inactivated
solution was subjected to PCR analysis. PCR was carried
out with the lacZ-specific primer lacZ2 Ž5X-TGCAGGA-
TATCCTGCTGATGAAGC-3X . and lacZ3R Ž5X-CATC-
CAGTGCAGGAGCTCGTTATC-3X . under the following
conditions; 30 cycles of 948C for 1 min, 558C for 1 min
and 728C for 2 min, and a final extension step of 728C for
7 min.
Inserted copy number and DNA rearrangements were
determined by Southern blot hybridization analysis using
the lacZ fragment as a probe.
4.4. Assay for b-galactosidase actiÕity in whole tissue
The tissue expression pattern of the transgene was
examined by b-galactosidase assay ŽFig. 1B.. Samples of
10 mg of tissue from transgenic mice were homogenized
rapidly in 2 ml of homogenization buffer Ž0.25 M Tris–
HCl, pH 8.0.. b-Galactosidase enzyme activity was mea-
sured in duplicate assays using ONPG as a substrate.
Seventy microliters of ONPG and 200 ml of cleavage
buffer containing b-mercaptoethanol were added to 30 ml
of tissue homogenized solution. After incubation at 378C
for 30 min, hydrolysis of ONPG to ONP anion produces a
bright yellow color with a peak absorbance at 420 nm that
can be quantified spectrophotometrically if b-Galactosi-
dase enzyme activity is present. One unit of enzyme
activity is equivalent to a conversion of 1 nmol of ONPG
at 378C.
4.5. Staining to detect b-galactosidase actiÕity on section
The procedure for staining consisted of fixation, sec-
tioning, and staining itself ŽFig. 1C.. The fixation and
sectioning steps took 12–24 h. The time of the staining
step was dependent on the strength of the promoter activ-
ity, and was generally achieved after overnight incubation.
Heart Spleen Lung Liver Kidney Adrenal gland
– – – – – –
– – – – – –
– – – – – 9.5 " 3.2
– – – – – –
– – – – – 11.3 " 3.2
162 E. Takahashi et al.r Brain Research Protocols 5 (2000) 159–166

Table 2
Expression of the transgene in various sites in the brain and adrenal gland of transgenic mice
Žq. Expression, Žy. no expression.
Abbreviations: St.s stratum, Mo.s molecular.
Lines Olfactory bulb Dorsal cortex Hippocampus Cerebellum Adrenal gland
St. oriens St. radiatum Pyramidal cell Mo. Granule cell Cortex Medulla
Wild-type y y y y y y y y y y
a 1A 6.3-lacZ-14 q q y y y y y q y y
a 1A 6.3-lacZ-15 q q y y q q q q y q
a 1A 6.3-lacZ-16 y y y y y y y y y y
a 1A 6.3-lacZ-30 q q y y q q q q y q

Transgenic mice were anesthetized with 10% nembutal,
and perfused transcardially with fixation solution. The
various tissues were removed, postfixed for an additional
30 min, immersed in 30% sucrose for 6–12 h, embedded
in OCT compound and sectioned with a cryostat at a
thickness of 15 mm. Two to three sections were collected
per silane-coated slide. Sections were incubated in X-gal
staining solution overnight in the dark at 378C. After
staining, the sections were rinsed with PBS for 3 min,
counterstained with Nuclear fast red for 3 min, rinsed with
PBS for 3 min, dehydrated through an ethanol series Ž70%,
80%, 90%, 100% for 3 min each. and Xylol for 3 min, and
mounted on coverglasses using Cedarwood oil. The
mounted sections are able to store at least 6 months
without discoloration in the dark at 248C
5. Results
We were interested in the molecular mechanisms under-
lying the neuron-specific expression of the mouse PrQ-
type Ca2q channel a 1A subunit gene. We have cloned the
5X-upstream region of the a 1A subunit gene w20x and
constructed a transgene carrying a 6.3-kb 5X-upstream re-
gion of the gene fused to the lacZ reporter gene w19x. Of a
total 400 injected and transferred eggs, we finally obtained
four transgenic founder mice identified by PCR and South-
ern blot hybridization analysis Ždata not shown.. All
founders transmitted the respective transgene and thus
lines were established.
To examine the tissue expression pattern of the trans-
gene, b-galactosidase assay was performed using protein
extracts from various tissues obtained from 10-week-old
F1 transgenic mice ŽTable 1.. Wild-type mice do not
express detectable levels of b-galactosidase in the brain,
heart, spleen, lung, liver, kidney or adrenal gland. On the
other hand, three of our four transgenic mouse lines ex-
pressed b-galactosidase activity in the brain, and two lines
expressed b-galactosidase in the adrenal gland. None of
these lines expressed detectable levels of b-galactosidase
activity in the heart, spleen, lung, liver or kidney.
Cellular expression pattern of the transgene in the brain
and adrenal gland of 10-week-old F1 transgenic mice was
examined by histochemical staining. Incubation of the
brain and adrenal gland sections from each transgenic
mouse line with X-gal generated blue staining in the cells
expressing the transgene ŽTable 2.. Wild-type mice exhib-
ited no positively stained cells in the brain or adrenal
gland. The levels and distributions of transgene expression
did not correlate with the copy number of transgene ŽTa-
bles 1 and 2., and no differences were observed in the
expression patterns of the transgene between males and
females in any of the transgenic mouse lines Ždata not
shown.. Three of four transgenic mouse lines were positive
for expression of b-galactosidase activity on sections of
the brain. Two lines expressed the transgene in the olfac-
tory bulb, dorsal cortex, hippocampus ŽFig. 2A. and cere-
bellum. In the hippocampus, transgene expression was
detected in the pyramidal cell layer in the CA area ŽFig.
2A,B., and in the molecular and granule cell layer in the
DG area ŽFig. 2A,C.. One line expressed the transgene in
the olfactory bulb, dorsal cortex and cerebellum, but not in
the hippocampus. Two of the four lines expressed the
transgene in the medulla of the adrenal gland, but not in
the cortex. The expression patterns and mechanisms of
regulation of the mouse PrQ-type Ca2q channel a 1A
subunit gene have been reported in detail elsewhere w19x.

Fig. 2. The expression pattern of the transgene detected by X-gal histochemical staining. Parasagittal sections were counterstained with Nuclear fast red.
The transgene was expressed in the pyramidal cell layer in CA, and in the molecular and granule cell layer in DG. ŽA. Hippocampus. Bars s 25 mm. ŽB.
Enlargement of upper inset. Bars s 80 mm. ŽC. Enlargement of lower inset. Bars s 80 mm. Abbreviations: CA s cornu ammonis, DG s dentate gyrus,
OR s stratum oriens, PY s pyramidal cell layer, RA s stratum radiatum, MO s molecular layer, GR s granule cell layer.
E. Takahashi et al.r Brain Research Protocols 5 (2000) 159–166 163
164 E. Takahashi et al.r Brain Research Protocols 5 (2000) 159–166
6. Discussion
6.1. Troubleshooting
6.1.1. Assay of b-galactosidase actiÕity in whole tissues
6.1.1.1. No color deÕelopment.
Ø The transgene is not in-frame.
Ø The transgene is too long to be completely translated
or to be active.
Ø The transgene does not contain sufficient regulatory
sequences for expression. This requires further regulatory
regions such as additional 5X-upstream sequence, or an
intragenic or a 3X-downstream region of the gene. In the
case of the tyrosine hydroxylase gene, authentic expression
was not induced by a 5X-upstream region alone, but re-
quired additional 5X-upstream, intragenic, and 3X-down-
stream regions w11x. Before starting transgenic experi-
ments, the promoter region should be tested in in vitro
transfection analysis. For the mouse PrQ-type Ca2q chan-
nel a 1A subunit gene, we tested promoter activity using
the placental alkaline phosphatase ŽPLAP. reporter gene,
which demonstrated that the 6.3-kb 5X-upstream region of
a 1A subunit gene used in transgenic analysis was sufficient
for expression in cultured cells w20x.
Ø The transgene contains signal sequences such as
those required for secretion due to the creation of new
regulatory sequences in construction of the transgene, and
resulting in secretion of the translated fusion protein. This
problem might be circumvented by use an internal riboso-
mal entry site ŽIRES. placed in front of the lacZ gene that
enables independent translation w1,22x.
Ø The transgene integration sites might reside in a
silent chromatin region. Several independent transgenic
lines should therefore be analyzed. Independent transgenic
lines are also needed to exclude any false interpretation
due to ectopic expression of the transgene or destruction of
the endogenous gene. In our study, the expression pattern
of b-galactosidase was consistent among transgenic mouse
lines despite variations in intensity of b-galactosidase ac-
tivity, suggesting that chromosomal effects could be ex-
cluded in these lines.
6.1.1.2. Background color deÕelopment.
Ø The incubation time should not be more than 30 min.
6.1.2. Staining to detect b-galactosidase actiÕity on sec-
tions
6.1.2.1. The tissue is cracked with holes.
Ø Insufficient cryo-protection.
Ø Repeated freezing–thawing during cryo-sectioning.
6.1.2.2. The sections detach from the slide during staining.
Ø The coating is not good.
6.1.2.3. Background staining.
Ø Background is due to the incomplete fixation, use of old
fixation solution, or inappropriate pH of fixation solution.
6.1.2.4. Crystals of X-gal deposited on the section.
Ø These may form upon prolonged staining as the staining
solution tends to age. In most cases, they may be washed
out with excess PBS. Minimum 3 = 20 min washes with
PBS are required before sections are dehydrated and
mounted on the slides.
6.2. AlternatiÕe and support protocols
Transcriptional mechanisms for the expression of a
gene have been analyzed by means of an in vitro transfec-
tion assay, which has been used to define the sequences
associated with cell-specific expression of genes. As tis-
sues are composed of highly heterogeneous cell popula-
tions with distinct functional features, it is important to
know the molecular mechanisms that regulate the temporal
and spatial expression patterns of a gene. However, due to
the limited availability of clonal cell lines, a few genes
have been successfully analyzed by in vitro analysis. To
circumvent these limitations and to verify the cis-regu-
latory elements working in vivo, transgenic mouse technol-
ogy is very useful.
Generation and analysis of transgenic mice carrying
promoter-reporter fusion transgenes are suitable and sim-
ple methods to define the importance of cis-regulatory
elements. The E. coli chloramphenicol acetyltransferase
ŽCAT. gene w13x, the luciferase gene w7x, the green fluores-
cent protein ŽGFP. gene w26x and the human growth hor-
mone ŽhGH. gene w14x have been used as reporter genes.
However, these reporter genes have some disadvantages.
The CAT, luciferase and GFP genes do not allow visual
detection of spatial expression without antibody or fluores-
cent microscopy, respectively. The hGH gene expressed at
high levels can have undesirable phenotypic effects on the
growth and development of transgenic mice. In this study,
we chose the E. coli lacZ gene as a reporter because of the
ease of enzyme assay and visualization on sections, and its
lack of influence on growth and development. The lacZ
gene has been used successfully in many cases to analyze
promoter activity in transgenic mice w2,6,8,9,21x.
It has been reported that the transgene includes a nu-
clear localization signal Žnls. at the N-terminus of the
lacZ-coding region w3,24x. However, the expression pattern
of lacZ does not differ regardless of the presence or
absence of the nls w10x. In our study, the transgene con-
tained no nls, and blue staining was detected not only in
the granule cell layer, but also in the molecular layer of the
 E. Takahashi et al.r Brain Research Protocols 5 (2000) 159–166
DG area. On the other hand, blue staining was detected in
the pyramidal cell layer, but not in the stratum oriens or
radiatum of the CA area. These results suggested that
localization of blue staining in different subsets of neurons
is controlled by distinct regulatory mechanisms. However,
these regulation mechanisms have yet to be characterized.
For immunohistochemical visualization on X-gal-stained
sections, use of a transgene including the nls might be
advantageous to obtain a clear positive of the cell types
expressing the transgene. Some papers on dual staining are
cited here as examples w10,18,23x.
In gene-targeting experiments, the expression pattern of
the targeted gene has generally not yet been characterized.
When lacZ is placed in frame with the endogenous gene,
the spatial and temporal expression pattern of lacZ allows
us to follow the normal expression pattern of the targeted
gene. Several gene targeting experiments using this strat-
egy have been reported w5,25x.
In gene trap experiments, lacZ has been used as re-
porter gene following transfection of murine embryonic
stem ŽES. cells w16,17x. Gene trap vectors can integrate
randomly into the host genome, but lacZ is activated only
when a correct integration within a transcriptionally active
endogenous gene has occurred. Due to transcriptional fu-
sion between the reporter gene and the target gene, the
lacZ expression pattern closely resembles at the target
gene. As the lacZ sequence is known, cloning of the target
gene can be readily achieved by generation of cDNA from
the lacZ fusion transcript using the 5X-RACE PCR method
and the corresponding gene can be further characterized.
More importantly, ES cells can be injected into eight-cell
stage embryos or blastocysts, b-galactosidase expression
can be monitored in chimeric mice, and mice can be bred
to homozygosity to identify possible phenotypic alterna-
tions caused by interruption of the gene, similarly to gene
targeting experiments.
In transplantation analysis, lacZ has advantages as an
intrinsic cellular maker allowing identification in the graft.
Although olfactory bulb transplantation is a well-char-
acterized model that has been widely used to study neu-
ronal plasticity and regeneration, it is insufficient as a
labeling method for graft identification. In a previous
study, an olfactory bulb graft was labeled with tritiated
thymidine w12x. However, this method has some disadvan-
tages, i.e. partial or insufficient labeling of a defined
neuronal subclass due to the length of their generation
time, and inability to follow the dendritic arborization and
axonal outgrowth of transplanted neurons. The drawbacks
of this labeling method can be circumvented by using the
olfactory bulb derived from transgenic mice expressing
lacZ as a donor. Using simple staining procedures for
b-galactosidase activity or immunocytochemistry with
anti-b-gal antibody, the donor neurons expressing lacZ
can be detected, and the donor neurons make contact with
recipient neurons after long-term survival can be con-
firmed w15x.
165
7. Quick procedure
1. The promoter region of the gene is cloned into the lacZ
gene vector.
2. Transgenic mice are produced by injection of linear
transgene into the male pronuclei of fertilized eggs.
3. Transgenic mice are identified by PCR or Southern blot
hybridization analysis from tail DNA biopsies.
4. The tissue expression pattern of the transgene can be
examined by assaying b-galactosidase activity.
5. Transgenic mice are perfused transcardially with fixa-
tion solution, and the tissue detected by the assay of
b-galactosidase activity is removed, sectioned and
stained with X-gal and Nuclear fast red. The stained
sections are dehydrated, covered with coverglasses and
examined using a microscope.
8. Essential literature references
Refs. w1,4,5,15,19,20x.
Acknowledgements
We thank Dr. M. Ohgoh for helpful advice.
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