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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

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochemistry 72: 248-254.

Etkin, L.D., and B. Fearman. 1987. Distribution, expression and germ-linetransmission of exogenous DNA sequences following microinjection intoXenopus laevis eggs. Development j)9_: 15-23.

Flytzanis, C.N., A.P. McMahon, B.R. Hough-Evans, K.S. Katula, R.J. Britten, andE.H. Davidson. 1985. Persistence and integration of cloned DNA inpostembryonic sea urchins. Developmental Biology 108: 431-442.

Gorman , CM., G.T. Merlino, M.C. Willingham, I. tastan, and B.H. Howard.1982a. The Rous sarcoma virus long terminal repeat is a strong promoterwhen introduced into a variety of eukaryotic cells by DNA-mediated trans-fection. Proc. Natl. Acad. Sci. USA 79: 6777-6781.

Gorman, CM., L.M. Moffat, and B.H. Howard. 1982b. Reccmbinant gencmes whichexpress chloramphenicol acetyltransferasa in mairmalian cells. Mol. Cell.Biol. 2: 1044-1051.

Hammer, R.E., R.L. Brinster, and R.D. felmiter. 1985. Use of gene transfer toincrease animal growth. Cold Spring Harbor Symp. Quant. Biol. ̂ 0_: 379-388.

Hippenmeyer, P.J., G.G. Krivi, and M.K. Highkin. 1988. Transfer and expressionof the bacterial NPT-II gene in chick embryos using a Schmidt-Ruppin retro-virus vector. Nucleic Acids Res. ̂ 6: 7619-7632.

Holtfreter, j . 1931. Uber die isolierter Teile des Amphibienkeimes. I I .Zuchtung von Keimen und Keimteilen in Salzlosung. Wilhelm Roux Archiv fuerEntwicklungsmechanik der Organismen 124: 404-466.

Hewlett, A.R., B. Cullen, M. Hertle, and M.J. Bissell. 1987. Tissue tropismand temporal expression of Rous-sarccma virus in embryonic avian limb inovo. Oncogene Res.. 1:: 255-263.

Jaenisch, R. 1988. Transgenic animals. Science 240: 1468-1474.

Lang, R.A., D. Metcalf, R.A. Cuthbertson, I. Lyons, E. Stanley, A. Kelso, G.Kannourakis, D.J. Williamson, G.K. Klintworth, T.J. Gonda, and A.R. Dunn.1987. Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF)develop accumulations of macrophages, blindness, and a fatal syndrome oftissue damage. Cell 51: 675-686.

Luciw, P.A., J.M. Bishop, H.E. Varmus, and M.R. Capecchi. 1983. Location andfunction of retroviral and SV-40 sequences that enhance biochemical trans-formation after microinjection of DNA. Cell 33.: 705-716.

Luciw, P.A., H. Opperman, J.M. Bishop, and H.E. Varmus. 1984. Integration andexpression of several molecular forms of Rous sarcoma virus DNA used fortransfection of mouse cells. Mole. Cell. Biol. A: 1260-1269.

Maclean, N., D. Penman, and Z. Zhu. 1987. Introduction of novel genes intofish. Bio-Technology 5_: 257-261.

Nerenberg, M., S.H. Hinrichs, R.K. Reynolds, G. Khoury, and G. Jay. 1987. Thetat gene of human t-lymphotropic virus typa I induces mesenchymal tumors intransgenic mice. Science 237: 1324-1329.

Overbeek, P.A., S.-P. Lai, K.R. Van Quill, and H. Westphal. Tissue-specificexpresion in transgenic mice of a fused gene containing RSV terminalsequences. Science 231: 1574-1577.

Dow

nloa

ded

by [

Uni

vers

ity o

f Fl

orid

a] a

t 13:

35 1

6 Fe

brua

ry 2

015

CAT MARKER GENE IN GOLDFISH 93

Purchase, H.G., and B.R. Burmester. 1978. Leukosis/sarcoma group. In:Hofstad, Calvek, Helmboldt, Reid, and Yoder (Eds). Diseases of Poultry,pp. 418-468. Iowa State University Press, Ames, Iowa.

Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: Alaboratory manual, Second Edition. Cold Spring Harbor Laboratory, ColdSpring Harbor, New York.

Schneider, J.F., E.M. Hallerman, S.J. Ycon, L. He, M.L. Cross, Z. Liu, A.J.Faras, P.B. Hackett, A.R. Kapuscinski, and K.S. Guise. 1989. Transfer o£the bovine growth hormone gene into northern pike, Esox lucius. J. Cell.Biochan. 13B: 173 (Abstract).

Simons, J.P., I. Wilmut, A.J. Clark, A.L. Archibald, J.O. Bishop, and R. Lathe.1988. Gene transfer into sheep. Bio/Technology 6} 179-183.

Small, J.A., G. Khoury, G. Jay, P.M. Howley, and G.A. Scangos. 1986. Earlyregions of JC virus and BK virus induce distinct and tissue-specific tumorsin transgenic mice. Proc. Natl. Acad. Sci., U.S.A. J33: 8288-2892.

Soriano, P., R.D. Cone, R.C. Mulligan, and R. Jaenisch. 1986. Tissue-specificand ectopic expression of genes introduced into transgenic mice by retro-viruses. Science 234; 1409-1413.

Southern, E.M. 1975. Detection of specific sequences among DNA fragmentsseparated by gel electrophoresis. J. Mol. Biol. 98: 503-517.

Stacy, N.E., A.F. Cook, and R.E. Peter. 1979. Spontaneous and gonadotrophin-induced ovulation in the goldfish, Carassius auratus L. Effects of exter-nal factors. J. Fish Biol. 15: 349-361.

Stuart, G.W., J.V. McMurray, and M. Westerfield. 1989. Replication, integra-tion and stable germ-line transmission of foreign sequences injected intoearly zebrafish embryos. Develop. 103: 403-412.

Svet-Moldavsky, G.J. 1958. Sarcoma in albino rats treated during the embryonicstage with Rous virus. Nature 182: 1452-1453.

Wilkie, T.M., R.L. Brinster, and R.D. Palmiter. 1986. Germline and somaticmosaicism in transgenic mice. Develop. Biol. 118: 9-18.

Yamamoto, T., B. deCrombrugghe, and I. Pastan. 1980. Identification of a func-tional pranoter in the long terminal repeat of Rous Sarcoma virus. Cell22: 787-797.

Yoon, S.J., E.M. Hallerman, M. Gross, 2. Liu, J.F. Schneider, A.J. Faras, A.R.Kapuscinski, and K.S. Guise. 1989. Transfer of the gene for neomycinresistance into goldfish, Carassius auratus. Aquaculture, in press.

Zhang, P., M. Hayat, C. Joyce, CM. Lin, L.J. Gonzalez-Villasenor, R. Dunham,T.T. Chen, and D.A. Powers. 1988. Gene transfer, expression, and inheri-tance of pRSV-trout-GH-cDNA in fish. First International Symposium onMarine Molecular Biotechnology 9-11 Oct., 1988, Baltimore, MD (Abstract).

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