by the Myosin Light Chain-2 Promoter and Developmental

629
Heart-Specific Targeting of Firefly Luciferase
by the Myosin Light Chain-2 Promoter and
Developmental Regulation in Transgenic Mice
Wolfgang-Michael Franz, Daniel Breves, Karin Klingel, Gottfried Brem,
Peter Hans Hofschneider, Reinhard Kandolf
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Based on hybridization studies indicating constitutive expression levels of the endogenous myosin light
chain-2 (MLC-2) gene in embryonic, fetal, and adult myocardium, a model system for selective targeting
of genes to the heart of transgenic mice has been developed. A 2.1-kb DNA fragment of the 5' flanking
region of the rat cardiac MLC-2 gene was fused to the firefly luciferase reporter gene and introduced into
fertilized mouse oocytes. In four independent transgenic mouse lines, the expression of the MLC-2luciferase fusion gene was found exclusively in heart muscle. In contrast to the endogenous MLC-2 gene,
no luciferase activity was detectable in slow-twitch skeletal muscle or any other tissue of transgenic mice.
This result suggests that the 2.1-kb DNA fragment of the 5' flanking region of the cardiac MLC-2 gene
contains the regulatory elements required for selective gene expression in cardiac myocytes in vivo. In
contrast to the endogenous steady-state MLC-2 expression during development, transgenic luciferase
activity was 10-fold higher during embryogenesis, when formation of the ventricular loop and septum
takes place. The enhanced luciferase activity in early heart development may suggest a growth-dependent
control mechanism, involving either transcriptional or posttranscriptional regulation. In conclusion, this
model system with the 2.1-kb ventricle-specific MLC-2 promoter sequence should facilitate the overexpression of gene products in the developing and mature heart muscle and further elucidate molecular
mechanisms of myocardial diseases such as cardiomyopathies. (Circ Res. 1993;73:629-638.)
KEY WoRDs * heart muscle * embryogenesis * gene expression * cardiomyopathy * mouse
model
C ardiomyopathies constitute a group of heart
muscle diseases that present both contractile
and electrophysiological abnormalities usually
leading to severe heart failure or sudden cardiac death.'
The majority of cases are diagnosed as "idiopathic
cardiomyopathies" because of their unknown etiology.
The challenge is to determine the various causes that
lead to cardiomyopathies, which are currently the focus
of both basic2-4 and clinical5 investigations. To study the
potential implications of exogenous and endogenous
genetic alterations in cardiomyopathies, an in vivo
model system that allows the selective expression of
foreign gene products in the normal and diseased heart
is necessary. Therefore, it is important to develop a
well-characterized transgenic animal model that specifically provides high-level transcriptional activity in the
heart muscle but little or no activity in any other tissue.
Striated muscle types can be functionally divided into
cardiac, slow skeletal, and fast skeletal by their expression of distinct patterns of tissue-specific protein isoforms, which include both myofibrillar proteins and
Received November 16, 1992; accepted June 29, 1993.
From the Department of Virus Research (W.-M.F., D.B., K.K.,
P.H.H., R.K.), Max-Planck-Institut fuir Biochemie, Martinsried,
FRG, and the Institut fur Tierzucht und Tierhygiene (G.B.),
Ludwig-Maximilians-Universitat, Munchen, FRG.
Correspondence to Dr Wolfgang-Michael Franz, Department of
Cardiology, Innere Medizin III, University of Heidelberg, Bergheimerstr. 58, D-69115 Heidelberg, FRG.
intracellular enzymes. The expression of many of these
muscle-specific proteins is developmentally regulated at
the level of transcription.6-9 Differentiation into cardiac
muscle leads to the activation of muscle-specific genes
encoding myosin heavy and light chains, a-cardiac and
skeletal actins, the troponins I, C, and T, tropomyosin,
and creatine kinase.10 Since cardiac muscle and other
excitable tissues share many electrophysiological, metabolic, and contractile properties, it is not surprising
that a defined subset of cardiac genes is coexpressed in
cardiac, skeletal, or neural tissues.7"1-'7 In contrast to
the myosin light chain-1 genes (MLC-1A, MLC-1V),
which are coexpressed and developmentally regulated
in cardiac and fast skeletal muscle tissue,18-20 no developmental studies have been conducted for the myosin
light chain-2 (MLC-2) gene.
Several isoforms of the phosphorylatable MLC-2
encoded by the cardiac/slow skeletal muscle, fast skeletal muscle, and smooth muscle/nonmuscle genes have
been identified.2' 23 The smooth muscle/nonmuscle
MLC-2 isoform is expressed in smooth muscle tissues
such as colon and uterus and in some, but not all,
nonmuscle cells. In contrast, it is not expressed in
skeletal and cardiac muscle tissues.21 Transgenic studies
of the rat fast skeletal MLC-2 gene have found cisacting sequences that will target expression specifically
in fast-twitch skeletal muscle cells,22 although the precise cis-regulatory elements required for fast-twitch
630
Circulation Research Vol 73, No 4 October 1993
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skeletal muscle expression have not yet been identified.
The cardiac MLC-2 gene has recently been shown to be
coexpressed at high levels, both in adult heart and
slow-twitch skeletal muscle.23 Comparison of the rat
cardiac with the rat fast skeletal MLC-2 genes reveals a
high degree of conservation.24 Each gene contains seven
exons of identical size with conservation of intron/exon
boundaries. However, the 5' flanking regions, which
presumably contain the main determinants for cellspecific regulation of gene expression, are widely divergent.25 Previous studies using rat and chicken cardiac
MLC-2 regulatory sequences have demonstrated successful reporter gene expression in primary cardiac
muscle but not in skeletal muscle cells.24,25
Since transcription rates of cardiac-specific genes are
greatly diminished in primary heart muscle cells and
since fundamental differences often exist between permanent in vivo expression in intact tissue and transient
in vitro expression in cultured cells,26 it is necessary to
investigate the activity of the 5' regulatory region of the
cardiac MLC-2 gene in vivo. Therefore, we set out to
establish transgenic lines containing cardiac-specific
promoters linked to the luciferase reporter gene that
could be used as a model system to study the selective
expression of foreign genes in normal and diseased
hearts. Transgene expression was then assessed in various cell types during both embryonic and fetal development. In four independent transgenic mouse lines,
the 2.1-kb control region of the cardiac MLC-2 gene
restricted expression of a luciferase reporter gene to
heart muscle tissue throughout development. In contrast to the endogenous MLC-2 gene, which is constitutively expressed in ventricles during embryogenesis and
coexpressed in slow-twitch skeletal muscle in adult
mice, luciferase activity was only found in heart muscle,
increasing during embryonic ventricular loop and septum formation and then gradually declining to a postnatal steady-state level. These results demonstrate that
the 2.1-kb MLC-2 promoter fragment behaves differently from the endogenous MLC-2 expression. Since
there is no coexpression in slow-twitch skeletal muscle,
it can be used to direct expression of foreign gene
products exclusively to cardiac muscle tissue from early
embryogenesis.
Materials and Methods
In Situ Hybridization Analysis
Embryos were frozen in liquid nitrogen and embedded
in Tissue-Tek OCT (Lab-Tek Products, Chicago, Ill).
Serial cryostat sections (6 gum) were prepared at -20°C
and mounted on microscopic slides that had been pretreated as previously described.27 Sections were fixed for
20 minutes in 4% paraformaldehyde/phosphate-buffered
saline (mmol/L: NaCl, 130; Na2HPO4, 7; and NaH2PO4,
3). Slides were then dehydrated through a series of
ethanol. Embryo sections were hybridized according to
established procedures28 with the following modifications: the hybridization mixture contained 100- to 200 -bp
fragments of a nick-translated mouse cardiac MLC-2
cDNA (200 ng/mL) derived from a full-length 645-bp
fragment.23 Probes were labeled with [a-35S]dATP and
[a-35S]dCTP (1200 Ci/mmol) to a specific activity of
1 x 108 cpm/,ug. Control probes were prepared from nonrecombinant vector DNA. Hybridization was performed
in 10 mmol/L Tris-HCl, pH 7.4, 50% (vol/vol) deionized
formamide, 600 mmol/L NaCI, 1 mmol/L EDTA, 0.02%
polyvinylpyrrolidone, 0.02% Ficoll, 0.05% bovine serum
albumin, 10% dextran sulfate, 10 mmol/L dithiothreitol,
denatured sonicated salmon sperm DNA (200 gug/mL),
and rabbit liver tRNA (100 ,ug/mL). Hybridization with
DNA probes was proceeded at 37°C for 36 hours. Slides
were then washed as previously described,28 followed by
incubation in 2 x standard saline citrate (SSC) for 1 hour
at 55°C. Slides were autoradiographed and stained with
hematoxylin/eosin.27
Slot Blot Analysis of Endogenous
MLC-2 mRNA Expression
To isolate total RNA, flash-frozen tissues were homogenized with an ultra turrax in a 4 mol/L guanidium
thiocyanate solution followed by phenol-chloroform extraction and propanol precipitation as previously described.29 Samples were solubilized in TES buffer containing 0.2% sodium dodecyl sulfate (SDS), 1 mmol/L
EDTA, and 10 mmol/L Tris-HCl (pH 7.5). After optical
density measurement, equal amounts of RNA (0.5 and
0.25 gtg) were deposited onto a sheet of nitrocellulose
using a filtration manifold (Minifold II, Schleicher &
Schuell, Inc, Keene, NH). Filters were baked in vacuo at
80°C for 2 hours and prehybridized at 42°C for 4 hours in
50% formamide, 2 x SSC, 120 mmol/L phosphate buffer
(pH 6.8), 20 mmol/L EDTA (pH 7.2), 0.2% SDS, 1%
sarcosyl, 4 x Denhardt's solution, and 250 1ug/mL denatured herring sperm DNA. Subsequently, the 645-bp
EcoRI mouse cardiac MLC-2 cDNA probe,23 labeled
with [a-32PJdCTP (3000 Ci/mmol) to a specific activity of
4 X 108 cpm/,ug by random priming,30 was added to the
prehybridization solution, and hybridization was carried
out overnight at 42°C. After posthybridization and washing under stringent conditions (2x SSC and 0.2% SDS at
50°C), filters were exposed overnight to an XAR-5 film
(Kodak). To ensure equal binding of total RNA to the
filters, the relative quantity of 18S RNA was determined
by rehybridization of the slot blots using an oligonucleotide probe (-ACGGTATCTGATCGTCTTCGAACC-)
labeled with [ry-32PIdATP (3000 Ci/mmol) to a specific
activity of 2x108 cpm/.tg by T4 kinase. The probe was
added to the prehybridization solution (5 x standard
saline phosphate EDTA, 5x Denhardt's solution, 0.5%
SDS, and 250 ,ug/mL denatured herring sperm DNA),
and hybridization was carried out overnight at 42°C.
After posthybridization (3x SSC at 42°C), filters were
exposed overnight to XAR-5 film (Kodak).
Generation and Breeding of Transgenic Mice
A 2.7-kb EcoRI DNA fragment of the 5' upstream
regulatory sequence derived from the rat cardiac
MLC-2 gene (Fig 1A) was coupled to the firefly luciferase reporter gene.25,31 Transgenic mice were generated using the construct diagrammed in Fig 1B. A 2.1-kb
MLC-2-luciferase fusion gene (2.1MLC-2-luc), derived
from plasmid pMLCLA5' (-2700 to + 12),25 was excised
at the Kpn I restriction sites as indicated. One picoliter
containing 500 to 1000 copies of the fusion gene in 10
mmol/L Tris-HCl (pH 7.5) and 0.2 mmol/L EDTA was
microinjected into male pronuclei of fertilized mouse
oocytes [(B6/D2)F1 x NMRI] according to established
procedures.32'33 A total of 341 injected eggs were transplanted into the oviducts of 17 pseudopregnant mice
Franz et al MLC-2-Luciferase Gene Expression in Transgenic Mice
(A)
a:
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(B)
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HF-2
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3' rat cardiac MLC-2 gene
HF-la HF-lb TATA
X-Luciferase-cDNA K
1
2. 1MLC-2-luc fusiongene
FIG 1. A shows the genomic map of the rat cardiac myosin light chain-2 (MLC-2) gene. Seven exons encode a 166-amino-acid
polypeptide. The 5' untranslated region contains the transcriptional start site, TATA box, HF-lb, HF-la, HF-2, and HF-3
elements,27 and the cardiac-specific sequence (CSS).25B shows the 2.1-kb MLC-2-luciferasefusion gene (2.1MLC-2-luc) usedfor
generation of transgenic mice. Restriction enzymes are indicated as follows: A, Ava II; E, EcoRI; and K, Kpn I.
MLC-2<2.lkb>
z
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(NMRI). DNA of transgenic founders and their offspring, obtained by backcrosses (Fl generation) or
breeding of siblings (F2 and F3 generation), was analyzed by Southern hybridization. For studies of ontogeny, homozygous F2 males of line FO 94 and FO 16 were
crossed with nontransgenic female mice. Gestational
age of intrauterine mouse embryos was related to the
first day of mucous plug formation of the pregnant
female, designated day 1 after conception.
Preparation and Analysis of Genomic DNA
High molecular weight DNA was isolated by incubating
1 cm of mouse tail in 0.5 mL HMWI buffer containing
(mmol/L) Tris-HCI, 10 (pH 7.5); NaCI, 150; and EDTA,
10; together with 0.4% SDS and 200 ,ug proteinase K
(Merck) for 12 hours at 37°C.34 DNA was phenol-chloroform-extracted, ethanol-precipitated, and resuspended in
30 gL TE buffer containing (mmol/L) Tris-HCl, 10 (pH
7.5) and EDTA, 1. Five micrograms of EcoRI-digested
DNA was electrophoresed and transferred to a nitrocellulose filter in 20 x SSC. Prehybridization buffer was used
as described above. Filters were hybridized with a
[a-"P]dCTP-labeled30 1.8-kb HindIll-Kpn I luciferase
fragment derived from pSV2/LA5'.31 After posthybridization and washing in 2x SSC, filters were exposed for 48
hours with enhancing screens at 80C.
-
Preparation of Tissue Samples for Luciferase Enzyme
Assay and Polymerase Chain Reaction
For analysis of luciferase expression, muscle tissues
such as heart, skeletal muscle (quadriceps femoris,
gastrocnemius, and soleus), smooth muscle (uterus),
and nonmuscle tissues such as gonads (testis or ovary),
lung, liver, spleen, kidney, thymus, salivary gland, brain,
skin, stomach, and intestines were removed from etheranesthesized transgenic mice and nontransgenic control
mice. For detailed localization of luciferase expression,
the myocardium was separated into atrial and ventricular tissue under a dissecting microscope. For developmental studies, the heart and the remaining thorax,
head region, abdomen, and tail region were dissected
and immediately flash-frozen in liquid nitrogen. Tissues
were then pulverized in liquid nitrogen and homogenized in 200 to 300 gL extraction buffer with 100
mmol/L KH2P04 (pH 7.8), 1 mmol/L dithiothreitol, and
0.1% Triton X-100. Tissue homogenates were instantly
refrozen in a dry-ice bath and lysed by thawing at 37°C
for 3 minutes. Cellular debris was pelleted for 15
minutes at 11 000g, and supernatants were stored at
-700C.
Total RNA was isolated from frozen tissues as described above.29 RNA samples from transgenic hearts
and muscle tissues were digested by RQ1-DNAse
(Promega Corp, Madison, Wis) in 50 mmol/L Tris-HCl
(pH 7.5) and 5 mmol/L MgCl2. After phenol-chloroform
extraction, RNA was reprecipitated and stored at
-800C.
Quantitative Luciferase Enzyme Assay
The luciferase assay was performed according to
established procedures31 with the following modifications: A 10-gL sample of protein extract was added to
100 ,aL of assay buffer containing (mmol/L) glycylglycine, 25 (pH 7.8); MgSO4, 10; EDTA, 2; and ATP, 2.5
(pH 7.5). The tube was placed in a monolight 2001
transilluminometer (Analytical Luminescence Laboratory, San Diego, Calif), and the reaction was initiated by
the automated injection of 100 guL of 1 mmol/L D-luciferin. Peak light emission of tissue luciferase activity
was recorded over a period of 10 seconds. Three
independent measurements were performed for each
tissue tested. Luciferase enzyme activity was expressed
in relative light units. Enzyme activities were related to
the amount of total cellular protein extract in milligrams, determined by the Bradford assay.35 For each
assay, a standard curve with gradually increasing
amounts of pure luciferase protein (Analytical Luminescence Laboratory) in 10 gg nontransgenic protein
was generated. By double logarithmic display, a regression line linear over a range of 4 logs was generated (see
Fig 2). Peak light emission was recorded over four
orders of magnitude in the linear range from 0.03 to 300
pg. The limit of detection of luciferase protein (molecular mass, 62 kD) was determined to be 30 fg or 2.9 x 105
molecules. Extrapolation of the light units per milligram
logarithm of positive transgenic hearts allowed quantification of the amount of luciferase expression per
milligram of total soluble protein. To relate the amount
of cardiac protein to 1 g of total heart, the following
transformation was performed. Approximately 5 mg of
protein derived from one transgenic mouse heart (total
Circulation Research Vol 73, No 4 October 1993
632
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1.5
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1.5
.5-2
7
4.5
5
6.5
3.5
4
5.5
6
log 10 of RLU or LU/mg proteln
FIG 2. Line graph shows the relation of luciferase protein in
picograms to the emission of light units (LU) per milligram
total protein. By double logarithmic display, a regression line
(y-1.102x-4.663) was generated. Peak light emission was
recorded over 4 logs in the linear range from 30 fg to 300 pg
ofluciferase. Extrapolation of the average L Uper milligram of
the four transgenic lines (8, 16, 18, and 94) allows quantitation of the level of luciferase expression. RLU indicates
relative LU; *, the average value of LU per amount of
luciferase standard; tthe average value of LU per milligram
protein of transgenic lines 94, 16, 8, and 18 from left to right.
2.5
3
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sWA
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weight, 100 mg) was solubilized in the extraction buffer,
which equals 50 mg of protein per l g of total heart.
Detection of Luciferase mRNA Expression by
Polymerase Chain Reaction
Two unique 23-bp oligonucleotide primers Luc-P2
(from +9 bp to +31 bp) and Luc-P3 (from +363 bp to
+387 bp) located within the luciferase coding region36
were synthesized. These primers amplified a 379-bp
fragment of firefly luciferase cDNA. For standard polymerase chain reaction (PCR) analysis, 2 gg of total
RNA was reverse-transcribed using the 3' antisense
primer, Luc-P3. One fourth of the reverse-transcribed
product was amplified by PCR using 1 gmol/L of each
primer in a reaction buffer containing 100 mmol/L
Tris-HCl (pH 8.3), 500 mmol/L KCl, 15 mmol/L MgC12,
0.01% (wt/vol) gelatin, and 200 gmol/L of each of the
four deoxynucleotide triphosphates.36 PCR conditions
included denaturation at 94°C for 45 seconds, annealing
at 64°C for 45 seconds, and polymerization at 72°C for 1
minute; after 40 cycles, an extension step of 7 minutes at
72°C was added. As a positive control, luciferase cDNA,
pSV2/LtA5', was transfected into cos cells.37 Non-reverse-transcribed RNA preparations were used to exclude genomic DNA contamination.
Results
Expression Pattern of the Endogenous Cardiac
MLC-2 Gene During Embryogenesis
Previous transgenic experiments have shown that
antigen presentation during early development confers
self-tolerance by the murine immune system, whereas
delayed appearance of the antigen results in an autoimmune response.38 Therefore, the early onset of gene
expression is an important prerequisite for a transgenic
model, which allows the introduction of foreign antigens
to the heart muscle. Since no data on prenatal expression of the endogenous cardiac MLC-2 gene have been
generated, we analyzed tissue specificity of MLC-2 gene
FIG 3. In situ hybridization analysis of cardiac myosin light
chain-2 (MLC-2) mRNA in a 13-day-old mouse embryo. A
shows saggital tissue section of a total mouse embryo hybridized with a [a- 35S]dATP/dCTP-labeled murine MLC-2
cDNA probe, demonstrating heart muscle-specific MLC-2
mRNA expression without coexpression in other tissues; B, in
situ positive ventricular tissue at a higher magnification
(autoradiographic silver grains are clearly localized in ventricular myocardial cells); and C, ventricular tissue of an embryonic heart hybridized with an [a- 35SdATP/dCTP-labeled
nonrecombinant plasmid control DNA. Sections were stained
with hematoxylin and eosin.
expression during embryogenesis. In situ hybridizations
using a 35S-labeled mouse cardiac MLC-2 eDNA probe
were performed to investigate the temporal and spatial
expression pattern of the endogenous cardiac MLC-2
gene during embryogenesis. Saggital sections of total
mouse embryos showed MLC-2 mRNA expression
throughout the entire section of the mouse ventricular
myocardium as displayed in Fig 3 for day 13 after
Franz et al MLC-2-Luciferase Gene Expression in Transgenic Mice
conception. Importantly, no expression was seen within
any other muscular or nonmuscular tissue (Fig 3A). As
illustrated in Fig 3B, autoradiographic labeling is clearly
restricted to ventricular myocardium, demonstrating
tissue specificity of MLC-2 expression during embryogenesis. No labeling of myocardial cells was observed
when mouse embryos were hybridized with the nonrecombinant vector control DNA probe (Fig 3C), demonstrating the specificity of in situ hybridization for detection of MLC-2 mRNA. These results indicate that the
endogenous MLC-2 gene expression is restricted to the
ventricular myocardium during embryogenesis. MLC-2
expression coincides with the education period of B and
T cells, which should result in a potential "self-acceptance" of transgenic proteins by the murine immune
system.
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Quantitative Analysis of the Endogenous Cardiac
MLC-2 Expression During Development
Quantitative changes in gene expression during cardiac development are a central feature of myocyte
differentiation. 0,39 So far, cardiac ventricular MLC-2
proteins have been shown to be constitutively expressed
after birth.411 To determine how the endogenous cardiac
MLC-2 gene is regulated during prenatal heart development, total RNA was extracted from embryonic,
fetal, newborn, and adult mouse hearts. Equal amounts
of total RNA were fixed to nitrocellulose and hybridized
with a 32P-labeled mouse cardiac MLC-2 eDNA probe.
As can be seen by autoradiography, a relatively homogenous signal was detected in prenatal, neonatal, and
adult heart samples (Fig 4). Rehybridization of the
filters using a 32P-labeled 1.8S oligonucleotide probe
confirmed equal amounts of RNA binding per slot.
Comparison of the cardiac MLC-2 transcripts with the
signals generated by the 18S RNA slots gave a constant
ratio. This demonstrates that, in relation to total RNA,
cardiac MLC-2 transcripts do not change during development. Thus, the endogenous MLC-2 promoter reveals a constitutive expression level during the entire
period of heart development studied. As a control, the
cardiac MLC-2 eDNA probe was hybridized with fastand slow-twitch skeletal muscle RNA. Specific hybridization was found with slow skeletal muscle RNA,
whereas fast-twitch skeletal muscle RNA gave no detectable signals. This result confirmed the coexpression
of the endogenous MLC-2 gene in adult mouse tissue of
cardiac and slow-twitch skeletal muscle as recently
published by Lee et al.23
Generation and Characterization of 2.JMLC-2-luc
Transgenic Mouse Lines
Since endogenous MLC-2 expression was ventricle
specific and occurred during the self-acceptance phase
of the murine immune system, the putative regulatory
promoter segment of the cardiac MLC-2 gene was
assessed for tissue-specific targeting of a reporter gene
in transgenic mice. A 2.1-kb 5' upstream MLC-2 DNA
fragment (Fig IA) was coupled to the firefly luciferase
reporter gene (Fig IB). To generate transgenic mice,
the 2.1MLC-2-luc fusion gene was microinjected into
the male pronuclei of fertilized mouse ooeytes. Inoculated zygotes were then implanted into pseudopregnant
females, and offspring were screened for transgene
integration. A total of 64 pups were born. Seven new-
MLC-2
18S
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A-FSk
A-SSk
FIG 4. Slot blot hybridization analysis of the cardiac myosin
light chain-2 (MLC-2) mRNA (left lane) at different time
points during heart development. Autoradiogram of the same
filter demonstrates equal binding of total RNA (0.5 pg) with
approximately the same amount of 18S RNA (right lane) from
different mouse heart tissues at the indicated ages of development (A indicates adult, N.B., newborn, and post concept.,
after conception). As a control, total RNAs of adult slowtwitch skeletal muscle (SSk) and adult fast-twitch skeletal
muscle (FSk) were hybridized with a 12P-labeled cardiac
MLC-2 cDNA probe.
born mice, designated FO 08, 11, 16, 18, 26, 47, and 94,
had the luciferase gene integrated into their genome, as
determined by Southern blot hybridization analysis
(data not shown). The seven positive founders (F0)
were mated with nontransgenic mice, and DNA analysis
of the 2.1MLC-2-luc offspring showed transmission of
the luciferase gene to the Fl generation. According to
an autosomal dominant trait, 46% of Fl descendants
(120 of 263) were carriers of the transgene. Inbred
crossings of hemizygous Fl mice (lines 16, 26, 47, and
94) gave rise to 79% 2.1MLC-2-luc F2 descendants (145
of 178), of which approximately 25% were homozygous
and 50% were hemizygous litters. Homozygous inbred
crossings of F2 mice (lines 16, 26, 47, and 94) generated
100% 2.1MLC-2-luc-positive progeny (135 of 135). Homozygous F3 mice were used to study MLC-2 promoter
activity in response to a double transgenic DNA dose.
Outbred crossings of homozygous F2 males produced
100% hemizygous offspring, which were used for subsequent analysis of luciferase expression at different
time points during development.
Heart-Specific Expression of the
2. JMLC-2-luc Transgene
Total cellular protein extracts of transgenic hearts
from 30- to 60-day-old hemizygous Fl mice, derived
from FO 08, 11, 16, 18, 26, 47, and 94, were tested for
luciferase enzyme activity. Four independent 2.1MLC2-luc lines (08, 16, 18, and 94) revealed positive lu-
634
Circulation Research Vol 73, No 4 October 1993
controt cheart>
heart
quadr.fem.muscle
soleus muscle
uterus
testis/ovary
W5
lung
0
liver
&M
C
p
FIG 5. Diagram of mean luciferase activities
(in light units [LU] per milligram protein) in
various organs (quadr.fem. indicates quadriceps femoris) of transgenic mice. Columns
represent average luciferase activity determined in the four 2.1-kb myosin light chain2-luciferase lines (8, 16, 18, and 94). From
each transgenic line, light activity was determined in at least 10 different positive F] mice.
r
pancreas
spleen
kidney
stomach
intestines
thymus
salivary gland
brain
skin
50000
100000
150000
200000
LU/mg protein
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ciferase activity ranging from 2.7 kLU/mg, where LU
indicates light units, (line 94) to 560 kLU/mg (line 18) of
soluble protein. Luciferase activities of Fl animals of
the two 2.1MLC-2-luc lines 08 and 16 have been contributed to the recent study of Lee et al.23 An average
light emission of 161 kLU/mg protein was registered in
transgenic hearts, which equals an absolute amount of
14 pg or 1.3x l O molecules of luciferase protein per
milligram of total heart extract. To relate luciferase
activity to the amount of luciferase molecules, light
emission was converted to protein concentration by
using the standard curve in Fig 2. Extrapolation of the
10 log of 560 kLU/mg of line 18 gave a maximal
luciferase protein expression level of 46 pg or 4.5 x 108
molecules per milligram of heart extract; the minimal
expression level calculated for line 94 equals 131 fg or
2.9x 106 molecules per milligram of cardiac protein. To
relate luciferase protein expression to 1 g of total heart,
a factor of 50x has to be included (see "Materials and
Methods"). Thus, the maximal amount of luciferase
expression equals 2.3 ng or 2.2x 10`I molecules and the
minimal amount equals 6.6 pg or 6.4x 107 molecules per
1 g of total heart tissue.
Extending luciferase expression analysis from hemizygous Fl mice to homozygous F3 mice, transgenic lines
94 and 16 were analyzed for their cardiac luciferase
activity. Heart extracts of 25 homozygous F3 animals
revealed 6.7 or 5.5 kLU/mg protein, which corresponds
to an average increase in luciferase enzyme activity of
80% to 90% in homozygous animals containing a double gene dose of the 2.1MLC-2-luc fusion gene. This
implies that luciferase gene expression from both alleles
takes place in an additive way. To complete luciferase
protein expression analysis, various transgenic and control tissues were tested for light activity. Average luciferase activities (LU per milligram protein) detected
in the four independent 2.IMLC-2-luc expressor lines
(8, 16, 18, and 94) are presented in Fig 5. Total cellular
protein extracts of fast skeletal muscle (eg, quadriceps
femoris), slow skeletal muscle (eg, soleus), and smooth
muscle (eg, uterus), as well as the nonmuscle tissues
listed, did not show any light activity above background.
Luciferase activity was detected exclusively in cardiac
tissue and predominantly in the ventricles (ratio of
ventricles to atria, 100:1) with an average activity of
161±65.5 kLU/mg (Fig 5).
To exclude the possibility of low-level expression of
luciferase in noncardiae muscle tissues of transgenic
mice, PCR analyses were performed. Hemizygous Ft
litters derived from high expressor line 18 were used for
mRNA expression analysis (Fig 6). After reverse transcription of total cellular RNA using a 3' antisense
luciferase primer (Luc-P3), cDNA was amplified by
PCR adding the 5' sense primer (Luc-P2). A specific
luciferase cDNA fragment of 379 bp was generated
from 2.1MLC-2-luc transgenic hearts only (Fig 6, lane
7). In contrast, no specific PCR product was amplified
from total eDNA of skeletal muscle, such as soleus, and
smooth muscle, such as uterus, even after 40 rounds of
amplification (Fig 6, lanes 8 and 9). These results
1 2 3 4 5 6 7 8 9
379 bp o
FIG 6. Agarose gel of luciferase-specific polymerase chain
reaction (PCR) products. The 379-bp band is the amplified
PCR product of oligonucleotide primers Luc-P2 and Luc-P3.
Lanes are as follows: lane 1, a 123-bp DNA ladder; lanes 2
through 4, PCRproducts of 100fg (lane 2), lOfg (lane 3), and
1 fg (lane 4) pSV2/LA'5 template; lanes 5 and 6, control
study with PCR amplification of reverse-transcribed mRNA
(lane 5) and non-reverse-transcribed mRNA (lane 6) from
pSV2!LA5'-transfected cos cells; and lanes 7 through 9,
reverse-transcribed total cellular RNA from 2.1-kb myosin
light chain-2-luciferase transgenic heart (lane 7), soleus
muscle (lane 8), and uterus (lane 9) from the F] generation of
transgenic line 18.
Franz et al MLC-2-Luciferase Gene Expression in Transgenic Mice
40000 -
635
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post-partum
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Age
FIG 7. Bar graph shows the developmental regulation of luciferase activity (in light units [LU] per milligram protein) in hearts
of hemizygous transgenic F3 mice between day 9 after conception (p.c.) and postpartum day 62. Columns represent average values
obtained from animals of transgenic lines 16 and 94. Prenatal values were obtained by pooling hearts of 8 to 12 embryos of one
litter. Each time point displays the average value obtained from three independent litters of the two transgenic lines. Postnatal values
were generated from individual hearts. Each time point shows the average value obtained from six hearts of the two transgenic lines.
demonstrate
a
selective expression of the luciferase
transgene in heart muscle tissue.
Expression of the 2.1MLC-2-luc Transgene During
Heart Development
To investigate prenatal expression activity from the
2.1MLC-2-luc fusion gene, hemizygous F3 progenies
from transgenic lines FO 94 and FO 16 were analyzed for
luciferase enzyme activity at different time points during embryonic, fetal, and postnatal development (Fig 7).
Embryonic heart protein extracts were generated as
early as day 9 after conception, when atrial trunk and
ventricular loop formation takes place. Until days 11
to 13 after conception, when the interventricular and
interatrial septum develops, luciferase expression levels
up to 36 kLU/mg protein were detected in the embryonic heart. From the beginning of fetogenesis at day
14.5 after conception, when the murine circulation
system is complete and functioning with an open foramen ovale, luciferase activity gradually declines until
postpartum day 15. A steady-state luciferase expression
level of 3.3 kLU/mg cardiac protein was observed from
postpartum days 15 to 62 (Fig 7). These results demonstrate that during the embryonic period of ventricular
loop and septum formation there is a 10-fold higher
luciferase activity compared with postpartum steadystate levels. In addition, enzymatic analyses of embryonic protein extracts from the remaining thorax, head,
abdomen, and tail and fetal protein extracts from tissues
as listed in Fig 5 did not reveal light activity at any time
point studied.
Discussion
On the basis of our finding of early endogenous
MLC-2 gene expression at constitutive levels in the
embryonic, fetal, and adult myocardium, a 5' regulatory
MLC-2 fragment was chosen to establish a transgenic
model for heart-specific gene expression. In the present
study, we have analyzed the ability of the rat cardiac
MLC-2 promoter to selectively target luciferase reporter gene expression to heart tissue of transgenic mice
during development. Different from the endogenous
cardiac/slow skeletal muscle MLC-2 gene expression,
our data demonstrate two properties of the 2.1-kb rat
cardiac MLC-2 sequence: (1) A constant cardiac muscle-restricted luciferase activity was observed postpartum in four transgenic lines. (2) In two lines, luciferase
activity was found to be 10-fold higher during the
embryonic period of ventricular loop and septum formation. The enhanced luciferase activity in early heart
development indicates a growth-dependent control
mechanism. Since no coexpression of the luciferase
reporter was detected in any other tissue except murine
myocardium, the 2.1-kb DNA fragment of the cardiac
MLC-2 gene should contain the regulatory elements
required for selective gene expression in cardiac myocytes in vivo.
Cardiac-Specific Gene Expression From the 21-kb
MLC-2 Promoter
Several activating and repressing elements responsible for cardiac-specific gene expression have been
mapped within the -2100-bp region of the MLC-2
promoter in tissue cultures.24'42-45 Five activating DNA
elements, HF-la, HF-lb, HF-2, HF-3, and the TATA
box, are located within a region from -198 to -19 bp of
the rat cardiac MLC-2 gene.42'43 A repressing element
called the cardiac-specific sequence (CSS) is located
further upstream between -1723 and -1686 bp of the
rat cardiac MLC-2 gene24 (Fig 1A). The HF-1 region, a
conserved 28-bp sequence between -72 and -45 bp,42
contains conserved sequences to known regulatory elements including CC(A+T-rich)GG (CArG) box/serum
responsive element (SRE)/element A,4546 AP-2 box,47'48
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and myocyte-specific enhancer-binding factor-2
(MEF-2) box/element B,4549 the latter being identical
with the HF-lb sequence.43 For muscle-specific transcription, only the proximal promoter consisting of
HF-lb and TATA box/element C is required.4t Addition of HF-la and HF-2 domains results in a musclespecific and inducible gene expression in transient tissue
culture assays.42,45 Since the nuclear CArG-, AP-2-, and
MEF-2-binding factors are found in differentiated cardiac as well as skeletal muscle cells,50 they may not be
sufficient to account for the heart muscle-specific activity of the MLC-2 promoter observed in transgenic mice.
However, specificity might be conferred by a unique
combination of factors or through binding of a cardiacspecific cofactor. Alternatively, repression of cardiac
MLC-2 promoter activity has been pointed to the 5'
flanking CSS element in primary fast-twitch and mouse
C2C12 skeletal muscle cell cultures.24 Removal of the
CSS sequence from the chicken cardiac MLC-2 promoter allowed transcription in fast-twitch skeletal muscle cells without affecting the transcriptional activity of
the promoter in cardiac muscle cells. CSS-binding proteins were identified in nuclear extracts from skeletal
muscle but not from cardiac muscle.24 A negative regulatory mechanism may therefore account for lack of
expression of the cardiac MLC-2 gene at least in fast
skeletal muscle tissue. However, it remains to be determined why endogenous cardiac MLC-2 mRNA is an
abundant transcript in both heart and slow-twitch (soleus) muscle of the adult rat and mouse23 and why
transgenic mice with a 2.1MLC-2-luc fusion gene display luciferase activity exclusively in cardiac muscle.
According to our results, the 2.1-kb MLC-2 promoter
contains all cis-regulatory elements required for restricted expression in embryonic and adult cardiac
muscle cells but obviously lacks critical sequences necessary for expression in slow skeletal muscle. This may
indicate that the cis-acting sequences required for expression in the slow skeletal context either lie further
upstream from the 2100-bp fragment or are located in
the downstream intronic portion of the gene. Therefore,
we suggest that distinct regulatory programs may have
developed for the expression of the cardiac MLC-2 gene
in heart and slow-twitch skeletal muscle.
Comparison of Endogenous and Transgenic Promoter
Activity During Cardiac Development
Expression of the cardiac MLC-2 mRNA and the
luciferase protein has been determined during ontogeny
of the mouse heart. The cardiac MLC-2 mRNA was
found to be constitutively expressed in relation to 18S
RNA during murine heart development. In contrast, a
10-fold increased luciferase protein synthesis driven by
the 2.1-kb MLC-2 promoter was detected during embryogenesis, when the ventricular loop and septum
formation takes place and the atrioventricular cushions
and the atrial septum with the remaining foramen ovale
appear. After completion of the fetal circulatory system
at day 14.5 after conception, a decrease in luciferase
activity was monitored. These findings extend our understanding of the developmental expression of the
cardiac MLC-2 gene; simultaneously, they raise the
question of a differential regulatory mechanism for luciferase reporter gene expression driven by the 2.1-kb
MLC-2 promoter. Little is known about the correlation
of MLC-2 mRNA and protein levels in the growing
myocardium. A single study performed during the development of chicken fast skeletal muscle reported that
MLC-2 protein levels correspond to the accumulation of
mRNA.i In the case of similar protein and mRNA
levels, it can be speculated that the 2.1-kb MLC-2
promoter region has lost negative regulatory elements,
which are necessary for a constitutive developmental
luciferase gene expression. On the other hand, a posttranscriptional mechanism such as a change in the rate of
protein synthesis could be possible. For example, the rate
of 18S and 28S rRNA transcription has been reported to
be upregulated in growing tissues.52 3 Since MLC-2
mRNA levels were related to 18S RNA, a simultaneous
increase can be postulated. On the other hand, it is
possible that the luciferase mRNA stability, rate of
transcription, and posttranscriptional mRNA processing,
which are important factors determining the rate of
protein synthesis, are changed in the growing myocardium. Therefore, more detailed analyses such as nuclear
run-on assays of luciferase and MLC-2 and Western blot
assays of MLC-2 protein during myocardial development
are necessary to resolve these questions.
Quantitative Analysis of the Luciferase Expression
Levels From the 2.lMLC-2-luc Transgene
For further cardiac gene expression studies, it is
important to estimate the strength of the 2.1-kb MLC-2
promoter activity. Therefore, the amount of luciferase
reporter enzyme in transgenic hearts was compared
with a representative tissue-specific household promoter and with the expected amount of MLC-2 mRNA
in heart muscle. A household promoter, such as phosphoglycerate kinase 2, revealed testis-specific luciferase
activity of 1600 LU/,g in transgenic mice.54 The maximal expression activity of the 2.1-kb MLC-2 promoter
falls with 560 LU/pgg into the range of household gene
expression.54
The concentration of total mRNA in heart muscle (R.
Kandolf, unpublished data) was estimated to be
21.3±+4.5 ,ug/g of total heart, which is close to the
reported concentration of mRNA in skeletal muscle of
26 ,ug/g tissue.55 The amount of MLC mRNA constitutes 2.0% of the poly(A) RNA.56 Since the ratio of
MLC-1 to MLC-2 is almost 1:1, approximately 1.0% or
213 ng of MLC-2 mRNA per 1 g of total heart can be
expected. Based on the assumption that the amount of
luciferase protein reflects the level of MLC-2 mRNA,
the highest luciferase expression in adult hemizygous Fl
mice (2.3 ng per gram of total adult heart) equals 1.1%
of the estimated MLC-2 mRNA. When it is taken into
account that the amount of MLC-2 mRNA expression
was reported in 16-day-old chicken embryos56 and the
luciferase protein expression was calculated for adult Fl
mice, a factor of 10x regarding the embryonic upregulation and a factor of 2 x regarding hemizygosity have to
be included. According to this calculation, luciferase
protein expression was estimated to be 22% of MLC-2
mRNA levels. This may suggest that most, if not all,
cis-acting elements for effective MLC-2 mRNA expression are located in the 2.1-kb regulatory sequence.
Implications for Cardiac-Specific Gene Expression
By the transgenic approach described in the present
study, other promoters with potential cardiac specificity,
Franz et al MLC-2-Luciferase Gene Expression in Transgenic Mice
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such as members of the contractile (a-myosin heavy
chain, ,B-myosin heavy chain, MLC-1A, MLC-1V, and
troponins C, T, and I), the ion channel (voltage-dependent Na+, Ca2+, and K' channels), the receptor (a- and
,3-adrenergic receptors), and the cardiac hormone (atrial natriuretic factor and renin) gene family, can be
characterized for their developmental, regional, and
quantitative expression activity. So far, cardiac-specific
promoter sequences tested (atrial natriuretic factor,
a-myosin heavy chain, and creatine kinase) show either
coexpression in skeletal muscle or other tissues.1057-59
To address a particular biochemical, physiological, or
pharmacological problem using a transgenic model, it is
necessary to know the detailed properties of the chosen
promoter sequence in order to distinguish tissue-specific influences from a systemic effect. The transgenic
model described in this study should provide a powerful
in vivo system for selective targeting of foreign gene
products to the ventricular myocardium. By this approach, viral gene regions of the cardiotropic coxsackievirus B3276061 are being expressed in a heart-specific
manner to unravel interference with myocyte function.
In addition, overexpression of oncogenes6263 and altered contractile proteins such as fB-myosin heavy
chain24 can be tested as potential pathogenes in the
etiology of cardiomyopathy. The transgenic model using
the ventricular myocyte-specific MLC-2 promoter will
therefore allow a genetic approach to understanding
basic molecular aspects of cardiac development and
regulation.
Acknowledgments
This study was supported by Grant Ka 593/2-2 from the
Deutsche Forschungsgemeinschaft, by Grant 321-7291-BCT0370 "Grundlagen und Anwendungen der Gentechnologie"
from the German Ministry for Research and Technology, and
by the Thyssen Foundation. We thank Dr K.R. Chien, University of California, San Diego, for providing us with the
MLC-2 promoter fragment that was used for these studies. We
are also grateful to Dr G Arnold for oligonucleotide synthesis
and to Dr W. Koch and Dr K.R. Chien for critical reading of
the manuscript. The excellent technical assistance of H. Riesemann is appreciated.
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Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and
developmental regulation in transgenic mice.
W M Franz, D Breves, K Klingel, G Brem, P H Hofschneider and R Kandolf
Circ Res. 1993;73:629-638
doi: 10.1161/01.RES.73.4.629
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