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Gene expression promoted by theRSV long terminal repeat element intransgenic goldfishEric M. Hallerman a f , John F. Schneider b , Mark Gross c ,Zhanjiang Liu d , Sung Joo Yoon a , Ling He c , Perry B. Hackett de , Anthony J. Faras b e , Anne R. Kapuscinski c & Kevin S. Guise ae
a Department of Animal Science , University of Minnesota , St.Paul, Minnesota, 55108b Department of Microbiology , University of Minnesota , St.Paul, Minnesota, 55108c Department of Fisheries and Wildlife , University of Minnesota ,St. Paul, Minnesota, 55108d Department of Genetics and Cell Biology , University ofMinnesota , St. Paul, Minnesota, 55108e Institute for Human Genetics , University of Minnesota , St.Paul, Minnesota, 55108f Department of Fisheries and Wildlife Sciences , VirginiaPolytechnic Institute and State University , Blacksburg, VA,24061Published online: 23 Sep 2009.
To cite this article: Eric M. Hallerman , John F. Schneider , Mark Gross , Zhanjiang Liu , Sung JooYoon , Ling He , Perry B. Hackett , Anthony J. Faras , Anne R. Kapuscinski & Kevin S. Guise (1990)Gene expression promoted by the RSV long terminal repeat element in transgenic goldfish, AnimalBiotechnology, 1:1, 79-93, DOI: 10.1080/10495399009525731
To link to this article: http://dx.doi.org/10.1080/10495399009525731
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ANIMAL BIOTECHNOLOGY, 1(1), 79-93 (1990)
GENE EXPRESSION PROMOTED BY THE RSV LONG TERMINAL
REPEAT ELEMENT IN TRANSGENIC GOLDFISH
Eric M. Hallerman1,6, John F. Schneider2, Mark Gross3, Zhanjiang Liu4,
Sung Joo Yoon1, Ling He3, Perry B. Hackett 4 , 5, Anthony J. Fara s 2 , 5 ,
Anne R. Kapuscinski3, and Kevin S. Guise1,5
Departments of 1Animal Science, 2Microbiology, 3Fisheries and Wildlife,
and 4Genetics and Cell Biology, and 5 I n s t i t u t e for Human Genetics, University
of Minnesota, St. Paul, Minnesota 55108
6Present Address: Department of Fisheries and Wildlife Sciences, Virginia
Polytechnic I n s t i t u t e and State University, Blacksburg, VA 24061
ABSTRACT
Persistence and levels of expression of the chloramphenicol acetyltrans-
ferase (CAT) marker gene under transcriptional regulation by the 5' long ter-
minal repeat element of the avian Rous sarcoma virus (RSV) were examined in
various tissues of transgenic goldfish, Carassius auratus. Evidence of the CAT
transgene was observed in 13 of 20 test individuals, with ten individuals being
apparent mosaics for the introduced construct. Above-background acetyltrans-
ferase activity was observed in tissues from 14 individuals, most frequently and
at highest levels in muscle. Acetyltransferase activities in muscle tissue of
transgenic individuals were as much as fifty times background. Transgene
expression, from the RSV promoter, observed in piscine muscle cells paralleled
earlier observations of RSV directed gene expression in avian and mammalian
systems.
79
Copyright © 1990 by Marcel Dekker, Inc.
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80 HALLERMAN ET AL.
INTRODUCTION
Successful alteration of an animal's phenotype through gene transfer
depends upon expression of the transgene product at biologically active levels
in the appropriate tissues and developmental stages of the animal. One major
factor affecting successful phenotypic alteration is the transcriptional pro-
moter element driving the expression of the transferred structural gene. The
long terminal repeat (LTR) element of the avian leukosis/sarcana retrovirus
Rous sarcoma virus (RSV) contains enhancer/promoter sequences regulating
transcription of the retroviral genome (Yamamoto et al., 1980; Luciw et al.,
1983), and has been used in expression vectors in gene transfer experiments
(Luciw et al., 1983, 1984). The tissue specificities and levels of gene
expression consequent to transfer of genetic constructs driven by the RSV
promoter have been characterized in chicken (Hewlett et al., 1987; Hippanmeyer
et a l . , 1988) and in mouse (Overbeek et a l . , 1986). Although RSV LTR-regulated
expression vectors have been introduced into carp (Zhang et a l . , 1988), goldfish
(Yoon et a l . , 1989), and Northern pike (Schneider et a l . , 1989), the tissue spe-
cif i c i t i e s and levels of expression promoted by the RSV LTR element have not
been characterized in piscine systems.
Following incorporation of a potentially useful promoter element and a
marker gene into an expression vector and screening for the transgene product in
a recipient species, the tissue specificity and levels of expression produced by
the regulatory element can thus be characterized (Jaenisch, 1988). Because
spawning of goldfish (Carassius auratus) i s easily induced (Stacy et a l . , 1979),
they provide an excellent piscine model system. In this study, transgene per-
sistence and levels of expression of the chloramphenicol acetyltransferase (CAT)
marker gene under the regulation of the RSV LTR element ware examined in several
tissues of goldfish hatched from eggs microinjected with the recanbinant DNA
METHODS
DMA construct. The pRSVcat construct incorporates the coding for the
chloramphenicol acetyltransferase protein fused t o the 51 long terminal repeat
of the Rous sarcoma virus (Gorman e t a l . , 1982b). I t s function was verified by
observation of antibiotic resistance in transformed E. coli cells.
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CAT MARKER GENE IN GOLDFISH 8 1
Production of transgenic fish. The circular pRSVcat construct was microin-
jected into the animal pole of newly-fertilized goldfish eggs (Yoon et al.,
1989). Microinjected (MI) eggs were allowed to develop in Holtfreter's solution
(Holtfreter, 1931) until the blastula stage, and in well water afterwards.
Goldfish were raised in aquaria for 413 days after hatching (denoted MI 1 to 6
in Table II) or for 394 days (denoted MI 7 to 20 in Table I I ) .
Persistance of DNA construct. High molecular weight DNA was purified
(Sambrook et al. , 1989) from the following tissues of the goldfish: muscle,
skin, spleen, liver, and gonad. Persistence of the DNA construct in the tissues
of the host was assayed by Southern hybridization (Southern, 1975) of EcoRl
digests using the 2533 bp EcoRl restriction fragment of the RSVcat construct as
the hybridization probe to detect presence of the transgene. The probe was
labelled with [32p] to a specific activity of 2 x 10^ c/ug by the random primer
method (Sambrook et al. , 1989), as par manufacturer's (Pharmacia), protocols.
Autoradiography was carried out for 7 days, using Kodak XAR-5 film and inten-
sifying screens.
CAT activity. Quantification of CAT enzyme activity followed Gorman et
al . (1982a,b). Briefly, 0.3 uCi [14C]-chloramphenicol (59.5 mCi/mM specific
activity) was incubated overnight (18 hr) with tissue homogenate. The reaction
mix was extracted with ethyl acetate, and the reaction products were separated
using thin layer chranatography. An autoradiograph was used to localize and
remove spots on the chrcmatogram corresponding to acetylated and unacetylated
[14C]-chloratnphenicol. The [14C] in the respective spots was quantified through
liquid scintillation counting. The concentration of protein in tissue homogena-
tes was quantified by using a Bio-Kad protein assay (BioRad, Richmond, CA) based
upon the protein-dye binding protocol of Bradford (1976). Binding of the dye to
protein was measured in terms of absorbance of light at 595 nm, and the con-
centration of protein was determined by comparison to a standard curve.
Specific activity of CAT in a given tissue sample was calculated as the number
of counts of acetylated [l^Cl-chloramphenicol divided by the number of
micrograms of total protein in the assay. Reported specific activities for
muscle and skin tissues are the mean values for two assays upon separately
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82 HALLERMAN ET AL.
TABLE ICHLORAMPHENICOL ACETYL TRANSFERASE ACTIVITIES IN SELECTED
TISSUES OF CONTROL GOLDFISH.
C represents raw counts par minute of [14C] acetylated chloramphenicol (xlO^).Assays were run for independent muscle and skin samples for some ind i v i d u a l s .
c/ug represents counts per minute of [l^C] acetylated chloramphenicol per ug oftissue. Results of Southern blot analysis have been suntnarized under DNA as -(no detection of DNA hybridization with RSV-LTR/cat probe); weak (minimaldetection of transgene through long term exposure); +, ++, and +++ (definitedetection of transgene, ranked by signal strength).
Individual
Cl
C2
C3
C4
C5
C6
Cc/(igCc/ugCc/ugC
cc/ugCc/ug
Muscle
21.562.68,43.172.7
238.772.5
Skin
3;152926; 4222421169.7
167.682.2
Brain
89.153.461.8
Liver
41.772.694.3
Spleen
125.2
145.4
145.4
Gonad
92.0
233251.1
dissected samples. Because the amount of tissue was limiting, reported specific
activities of other tissues represent the results of a single assay. The CAT
activity associated with a given tissue sample was considered to be above
background if i t was twice the highest valus observed among control samples
drawn from the respective tissues.
RESULTS
Background CAT activities in goldfish tissues. Despite earlier reports
that eukayotic cells exhibit no endogenous CAT activity (Gorman et al., 1982b),
we did observe a low-level background of acetylated chloramphenicol in control
fish tissue horregenates (individuals C-l to C-6, Table I ) . Goldfish cells may
indeed lack specific CAT activity, but with the long incubations (18 hr) used in
this study, activities presumably of non-specific acetyltransferases reached
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2>pa7;
6 7 M
B L S p G M S k A B L S p G M S k B L G M S k B L S p G M S k B L S p G M X S k B M S k B M
9 10 11 12 13 14 15 16 17 18 19
M S k B M S k O M S k O X B M S k O M S k O B M B M S k B M *• S k O B M S k O M S k O B B M S k
TFIGURE 1.
Southern analysis of DNA frcm specified tissues of microinjected goldfish,digested with EcoRI, and hybridized with [32P]-labelled RSV-LTR/CATconstruct. M = muscle, S = skin, B = brain, L = liver, N = spleen, G =gonad, and 0 = pooled organs.
oo
00
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84 HALLERMAN ET AL.
TABLE I ICHLORAMPHENICOL ACETYLTRANSFERASE ACTIVITY AND PERSISTANCE OF THE pRSVCAT
CONSTRUCT IN SELECTED TISSUES OF GOLDFISH HATCHED FROMpRSVCAT MICROINJECTED EGGS.
C represents raw counts per minute for [l^C] acetylated chloramphenicolAssays were run for independent samples of muscle and skin.
c/ug represents counts par minute of [l^C] acetylated chloramphenicol per ug oft i s s u e . Results of Southern b l o t analyses have been sunmarized as in Table I .
N.A. — Not available due t o li m i t i n g amount of t i s s u e .
Individual
MI-1
MI-2
MI-3
MI-4
MI-5
MI-6
MI-7
MI-8
MI-9
MI-10
Cc/ugDNACc/ugDNACc/ugDNACc/ugDNACc/ugDNACc/ugDNACc/ugDNACc/ugDNACc/ugDNACc/ugDNA
Muscle
160;8264+
892;593300
-14;612130++
5;610Weak
566;145230++
556;97160
-598;921490Weak
177;790250Weak
1;459.2-
246;1264+
Skin
10; 2228+
15; 1042
f12; 1414-
11; 713+
31;720-H-+
18; 1718-
39; 1435-
6; 3218+
16; 612-
2;510+
Brain
430-
32.2-
N.A.-63.3-
210190+ft-6567-
18190N.A.147.4-46.0-72.7N.A.
Liver
10.7-
11.0-43.6-
31.2-
12.4-H-28.6N.A.
Spleen
62.7-75.3-65.7-53.4-
31.0Weak22.3N.A.
Gonad
38.6Weak3
25.0—6
16.0f
1926.0Weak6
150.0+5
59.0N.A.
RxDledOrgans
109.7N.A.
104.7-
32.0-
40120
+
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CAT MARKER GENE IN GOLDFISH 85
TABIE I I (cont'd)
PooledIndividual Muscle Skin Brain Liver Spleen Gonad Organs
21.7
41.6
32.2
N.A.20.8
10.3+++41.6
42.4
N.A.33.4
N.A.73.3
N.A.31.6
MI-11
MI-12
MI-13
MI-14
MI-15
MI-16
MI-17
MI-18
MI-19
MI-20
Cc/ngDNACc/ngDNACc/ugDNACc/pgDNACc./ugDNACc/ugDNACc/ugDNACc/^igDNACc/ugDNA
cc/ugDNA
226;1059-
274;944210
+132,-922390
-57;916-
21;910+f+
55; 1549Weak14; 116.4
Weak2;63.4N.A.68;14571-
29;10072Weak
21; 57.6—
14; 5217+
11;654560N.A.2;1114-
8; 179.3-H-+
5;53.4-
3;82.9-
2;713N.A.3;65.4-
5;66.5_
512—
2038N.A.3076-41.9N.A.97.1Weak119.8-52.5-
227.4—52.7-4
11—
observable threshholds. Such background CAT activities were generally less than
10 counts/ug protein. Higher background CAT activities were observed in skin
(individuals C-l, C-2, and C-3) and in gonad and muscle (individual C-5).
Persistence of pRSVcat. Screening of Southern blots of DNA samples pre-
pared from tissues of 20 test goldfish (Figure 1, Table II) indicated the per-
sistence of the pRSVcat construct within certain tissues. The CAT gene probe
hybridized to high molecular weight DNA samples from 11 of the 20 test indivi-
duals. Hybridization alone could not rule out the possibility of extrachromoso-
mal persistence of the introduced DNA construct (Maclean et a l . , 1987).
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86 HALLERMAN ET AL.
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•
•
. * *
• * • • •
f tttttttt??•*? tftf ttttfff • tfffttmtfftf"..f
>
oz
oor1
tttttttt:2 '.2 n 13 n n 14M P P K S S M
Cl 14 15 15 V> 10 16 10 17 17s P P M r s M ;> :i M
C2 17 IB IS IS 19 19 19 20 20M r s M i' s M P S M
ttftttttttttt•i '1 C I, 1! 1 1 M f-
FIGURE 2. Autotradiographs of thin layer chramatographic analyses of CAT activity in tissues fran RSV-LTR/catmicroinjected (MI) and control (C) goldfish. M = muscle, S = skin, B = brain, L = liver , N = spleen, G = gonad,and P = pooled organs.
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88 HALLERMAN ET AL.
Hybridization to DNAs prepared from the various tissues of nine individuals
included both positive and negative results (Table I I ) . Both negative and
strongly positive hybridizations were observed in certain individuals (e.g.,
individual MI-9). The observations of presence and absence of hybridization to
DNAs from various tissues of an individual indicated mosaicism of the introduced
construct. Three individuals (MI-5, MI-10, and MI-15) showed presence of the
transgene in a l l analyzed tissues. Since not a l l tissues were analyzed, even
these three individuals may be mosaics. Ten individuals (MI-1, MI-2, MI-3,
MI-4, MI-7, MI-8, MI-12, MI-16, MI-17, and MI-20) showed varying degrees of
mosaicism fran fish that were strongly positive for the transgene in some
tissues and negative in others (MI-3) to individuals that were weakly positive
for the transgene in only one analyzed tissue (MI-2, MI-7, MI-10, MI-17, MI-20).
CAT activity in goldfish tissues. Tissues from six control (Table I) and
20 fish hatched front microinjected eggs (Table II) were assayed for CAT activity
(Figure 1). Fourteen test individuals bore at least one tissue exhibiting CAT
activity of a level at least twice that observed in the corresponding tissue of
any control fish. The distribution of elevated levels of CAT activity suggested
some tissue specificity of expression.
—Muscle. Fourteen individuals exhibited markedly elevated CAT activities
ranging fran 7-54 tines background (2.2 cpn/ug) (MI-1, MI-2, MI-3, MI-5,
MI-6, MI-7, MI-8, MI-10, MI-11, MI-12, MI-13, MI-16, MI-19, and MI-20).
Eight individuals exhibiting elevated CAT activity in muscle also showed
CAT activity in other tissues.
—Skin. Only MI-13 exhibited elevated CAT activity, with a value twenty
times higher than background (15.5 cpn/ug).
—Brain. CAT activities markedly greater than controls were observed in
brain tissue samples of six individuals (MI-1, MI-5, MI-6, MI-7, MI-12,
and MI-13), ranging fran 6-110 tines background (4.8 cpn/ug).
—Liver. No CAT activities above background level (2.9 cpn/ug) were
observed.
—Spleen. No CAT activities above background level (5.3 cpn/ug) were
observed.
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CAT MARKER GENE IN GOLDFISH 89
—Gonads. Notable variability in background CAT activity among controls
was attributable to sexual differentiation. Well developed gonads
appeared to show higher protein levels, and hence lower specific activi-
t i e s , than less developed gonads. Among individuals MI-1 to MI-6 (Table
I I ) , gonadal development had occurred, and sexual differentiation could
be observed. Among these individuals, above-background level (12 cpn/|ig)
of CAT activity were evident in at least one individual (MI-5).
Among smaller individuals (MI-7 to MI-20), CAT activity assays were carried
out upon samples of a l l organs collectively. In only one case, MI-10, was ele-
vated CAT activity observed. This activity could not be associated with any
particular tissue.
The data may be subdivided into four groups based on the presence or
absence of detectable DNA versus the expression of the CAT transgene. The
expression of the CAT gene in the presence of transgene DNA, and the lack of CAT
gene expression in the absence of transgene DNA are self explanatory. The lack
of CAT gene expression in the presence of transgene DNA may represent simply the
persistance of an incomplete transgene. The opposite circumstance, the obser-
vation of CAT expression in the absence of detectible transgene DNA is not
without precedent. The origin of the discordancy may l i e in the relative sen-
sitivity of CAT versus DNA analyses. Detectable levels of expression might
occur in a subset of the cells in a tissue, while a single copy of a plasmid in
the same subset could prove undetectable (Wilkie et a l . , 1986).
DISCUSSION
Mosaicism of Transgene Activity. In the transgenic goldfish exa-
mined in this study, the highest levels of RSV-induced transgene activity were
observed in muscle, with lesser levels of activity sometimes observed in brain
and gonad. Others have shown in mammals (Gorman et al., 1982a; Overbeek et al. ,
1986) that the amount of CAT-specific rriRNA encoded by pRSVcat correlates with
the level of CAT enzyme activity. Assuming that the CAT mRNA and CAT protein
are equally stable in a l l tissues, the expression of the CAT transgene under the
regulation of the RSV LTR observed in transgenic goldfish in this study is
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90 HALLERMAN ET AL.
consistant with previous observations in tissues of adult transgenic mice
(Overbeek et al., 1986) and chicken embryos (Howlett et a l . , 1987; Hippenmeyer
et al., 1988) where the RSV LTR is active in muscle cells.
The observed expression of RSV LTR-regulated transgenes in tissues of meso-
dermal origin parallels the observation of tumorigenesis in such tissues
following RSV infection in chicken (Purchase and Burmester, 1978) and mouse
(Svet-Moldavsky, 1958). The molecular basis for this tropism has been linked to
response by the viral regulatory elements to tissue-specific stimuli (Overbeek
et al., 1986), the precise nature of which is uncharacterized. Although
LTR-induced transgene expression was generally observed in tissues normally
transformed by RSV, the observation of ectopic expression is not without prece-
dent, having been observed in mice (Soriano et a l . , 1986; Small et al . , 1986;
Nerenberg et al., 1987). Ectopic expression in some individuals has been
hypothesized to be a consequence of the site of gencmic integration (Soriano et
a l . , 1986; Lang et al. , 1987).
The detection of transgene in some but not a l l tissues of an individual
fish suggests probable mosaicism for the pRSVcat construct. Mosaicism for
introduced DNA constructs in a wide range of animals (Flytzanis et a l . , 1985;
Wilkie et a l . , 1986; Etkin and Pearman, 1987; Stuart et a l . , 1988; Simons et
al., 1988) has been attributed to delay in the integration of the transgene
construct into host genomic DNA. This was probably the case among certain of
the transgenic goldfish examined in this study. The Southern analysis of these
fish suggest that much of the DNA detected is either unintegrated or integrated
as tandem copies. Delayed integration may also be the cause of the widely
disparate gene expression seen in muscle and skin samples from several indivi-
duals (MI-3, Ml-6, MI-10, andMI-13). In these individuals, a large difference
was detected in CAT activities between two separate tissue samples derived from
the same tissue of an individual. I t is possible, that our randan sampling
detected patches of tissue that were derived from different clonal lineages. If
the transgene were lost or modified early in the development of a clonal line,
then the-resultant tissue could display a patchy expression of the transgene,
much as a calico cat displays coloration patches due to random X inactivation
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CAT MARKER GENE IN GOLDFISH 9 1
during development. Biological activity of introduced constructs has been
observed in t a i l tissues of transgenic mice where analysis of t a i l DNA revealed
no integrant (Hairmer et al., 1985). Thus detection of transgenesis in the
microinjected (Go) individual requires assay of multiple tissues to counter the
complication of mosaicism, and should entail both analyses for transgene detec-
tion (Southern) and gene expression. Only in this manner may a more accurate
picture of the degree of success achieved in production of a transgenic animal
be obtained.
Practical Aspects of Findings. The elevated level of expression in muscle
tissue of CAT transgenes promoted by the RSV LTR element suggests i t s u t i l i t y in
expression vectors intended for practical genetic engineering applications. For
example, fast-growth of transgenic animals might result from elevated levels of
expression of growth factors in muscle tissue. This hypothesis is supported by
preliminary results from two experiments where RSV-LTR/growth hormone constructs
were introduced into fish. Accelerated growth was observed among northern pike
bearing an introduced RSV-LTR/bovine growth hormone construct (Schneider et al.,
1989) and among coimon carp bearing an introduced RSV-LTR/rainbow trout growth
hormone construct (Zhang et al., 1988).
ACKNOWLEDGEMENTS
The authors g r a t e f u l l y acknowledge t h e cooperation of Mite Voss. This work
was supported i n p a r t by g r a n t s from t h e L e g i s l a t i v e Carmission on Minnesota
Resources of the S t a t e of Minnesota ( t o authors A.J.F., P.B.H., A.R.K. and
K.S.G.), and by th e Minnesota A g r i c u l t u r a l Experiment S t a t i o n (A.R.K. and
K.S.G.). This i s c o n t r i b u t i o n number 17,622 of t h e Minnesota A g r i c u l t u r a l
Experiment Station Scientific Journal Article Series. This work is the result
of sponsorship by the Minnesota Sea Grant College Program under Grant R/3 and
R/4 (to K.S.G. and P.B.H.), and is research contribution no. 253. The Sea
Grant College Program is supported by the NOAA Office of Sea Grant, Department
of Commerce, under Grant No. NQAA-86AA-D-SG112-01. The U.S. Government is
authorized to reproduce and distribute reprints for government purposes, not
withstanding any copyright notation that may appear hereon.
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92 HALLERMAN ET AL.
LITERATURE CITED
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