A vector for expressing foreign genes in the brains and hearts of transgenic mice

ELSEVIER

Genetic Analysis: Biomolecular Engineering 13 (1996) 159 163

NENETIC ALYSIS BlomolecularEngineering

A vector for expressing foreign genes in the brains and hearts of transgenic mice David R. Borchelt a'*, Janine Davis% Marek Fischeff, Micheal K. Lee a, Hilda H. Slunt d, Tamara Ratovitsky J, Jean Regard d, Neal G. Copeland f, Nancy A. Jenkins f, Sangram S. Sisodia a,b, Donald L. Price ~,b,c,d aDepartment of Pathology, Johns Hopkins School of Medicine, 720 Rutland Ave., 558 Ross Building, Baltimore, MD 21205-2196, USA bDepartment of Neuroscience, Johns Hopkins School of Medicine, 720 Rutland Ave., 558 Ross Building, Baltimore, MD 21205-2196, USA ~Department of Neurology, Johns Hopkins School of Medicine, 720 Rutland Ave., 558 Ross Building, Baltimore, MD 21205-2196, USA aNeuropathology Laboratory, Johns Hopkins School of Medicine, 720 Rutland Ave., 558 Ross Building, Baltimore, MD 21205-2196, USA eInstitute for Molecular Biology, University of Zurich, Abteilung I, Honggerberg, Zurich 8093, Switzerland fMammalian Genetics Lab6ratory, ABL-Basic Research Program, NCI-Frederick Cancer Center Research and Development, Frederick, MD 21702, USA Received 20 May 1996; revised 21 October 1996; accepted 28 October 1996

Abstract

An expression plasmid (MoPrP.Xho), for use in transgenic mice, was developed from the promoter, 5' intronic, and Y untranslated sequences of the murine prion protein gene. Analyses of mice harboring the MoPrP.Xho construct with cDNA genes encoding the amyloid precur,;or protein (APP) and human presenilin 1 demonstrated that this vector provides relatively high levels of transgene-encoded polypeptides in brains and hearts of transgenic mice. The MoPrP.Xho vector should be very useful in strategies designed to overexpress a variety of wild-type and disease related mutant transgenes in the heart and brain. © 1996 Elsevier Science B.V. All rights reserved

Keywords: Expression vector; Transgenic; Brain

1. Introduction

Achieving elevated levels of transgene product is often a critical step in modeling autosomal dominant human neurodegenerative diseases in transgenic mice [1]. In investigations of the human prion diseases, the mammalian prion protein (PrP) gene was recognized as a vehicle to express foreign proteins in the central and peripheral nervous systems [1-3]. Initial investigations demonstrated that the coding sequences of a 42 kb cosmid clone of the Syrian hamster PrP gene, which encodes the entire open reading frame in a single exon

* Corresponding author. E-mail: [email protected] 1050-3862/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S 1 0 5 0 - 3 8 6 2 ( 9 6 ) 0 0 1 6 7 - 2

[4], could be exchanged for PrP c D N A genes from other species [2,3]. Subsequently, a vector derived from the Syrian hamster PrP gene (cosSHaPrP.neo) [2] was used to express recombinant amyloid precursor proteins in transgenic mice [1]. Although efficacious, the cosSHaPrP.neo vector cannot accept large c D N A genes because the upper limit of cosmids is approximately 45 kb [5]. However, one of us (M. Fischer) recently demonstrated that a re-engineered murine PrP genomic fragment (phgPrP) in which the second 10 kb intron and 15 kb of the 3' untranslated sequence (Fig. 1C) were removed, expressed PrP in an identical pattern as a full-length genomic clone (cos6.I/LnJ-4), with the exception that phgPrP transgenes were not expressed in Purkinje cells of the cerebellum [6].

160

D.R. Borchelt et al. / Genetic Analys&: Biomolecular Engineering 13 (1996) 159-163

2. Materials

and methods

Based on the past success with the Syrian hamster PrP cosmid vectors [1-3], we recognized the potential utility of a smaller vector, derived from the re-engineered murine PrP genomic fragment (phgPrP), in studies to express large cDNAs. In the present study, we replaced the PrP open reading frame in the phgPrP plasmid with a unique Xho I restriction endonuclease site (Fig. 2A-C). Subsequently, we inserted a cDNA A

E1 cos6EMBL SHaPrP H

B

E1 E2 PrP cos6.11 MoPrP , ~ , ~ ]

C

phgPrP MoPrP

I

5 kb

I

I

I

PrP

El E2-PrP

I

I

I

I

i

I

I

I

I

Fig. 1. Structure of genomic Syrian hamster and murine PrP genes contained within cosmid or plasmid clones (cos6EMBL SHaPrP map reproduced from Basler et al. [4] and Scott et al. [2]; cos6.I/LnJ-4 MoPrP map reproduced from Westaway et al. [19]; phgPrP Mo PrP map reproduced from Fischer et al. [6]). El, exon 1; E2, exon 2; PrP, exon 3 containing the PrP open reading frame. Each division on the line scale equals 5 kb of sequence.

Not I

B

II

ph~PrP

A I

Q2-+2[+E~c2 I C~cB

LineTransgenename Kpn I

Not I I

encoding a murine 695 amino acid isoform of the amyloid precursor protein (APP) possessing a 'humanized' Aft domain and mutations (K595N; M596L) that are linked to familial Alzheimer's disease [7]. Following standard pronuclear injection of DNA into C57BL/ 6J x C3H/HeJ F2 embryos, three lines of mice were generated which possessed between 3 and 10 copies of the transgene as measured by Southern blot analysis (not shown). In the brains of transgenic progeny, the level of accumulated APP, relative to nontransgenic littermates, was elevated by 2-3 fold (Fig. 3A). To confirm expression of the transgene-encoded polypeptide, immunoblot analyses were performed with a monoclonal antibody (6El0) that recognizes sequences unique to the human Aft region [8,9] (Fig. 3B). In all three lines of transgenic mice, transgene-derived APP was also present at high levels in heart, but was below the level of detection in the liver, lung, and kidney (Fig. 4). Because APP is a ubiquitously expressed protein [10-12], it is unlikely that the lack of transgene expression in liver, lung, and kidney is due to polypeptide instability and we interpret our findings to suggest that transgene expression in these tissues is substantially lower.

Nar I

1

213

4

516

7

8

A

phgPrP.Not I KpnI-XhoI Nat I

C

PCR product

D

Kpnl-XhoI Nar 1 II Not I ~ ~! I

Not I I ¢'q

--97 Not I

I I

MoPrP.Xho I

I

I

I

I

I

I

I

I

I

I

--68 I

I

tkb Fig. 2. Synthesis of the expression plasmid MoPrP.Xho. (A) The map of phgPrP genomic fragment in a pBluescript KS + plasmid. Boxes, exonic sequences; open box, noncoding 5' sequences; black box, coding sequences; cross-hatched box, noncoding 3' sequences. The Nar I site is not unique. (B) The initial modification to phgPrP was to remove the 3' Sal I site and replace it with a Not I site, facilitating the excision of plasmid sequences prior to microinjection. The phgPrP plasmid was digested with Sal I, filled in with a Klenow fragment, ligated with Not 1 linkers, then digested with Not I and cloned into pBluescript in which the Xho i site had been destroyed. (C) Subsequently, the 3' untranslated sequences were amplified by PCR, using oligonucleotide primers (arrows) complementary to sequences in pBluescript (antisense) and sequences just downstream of the open reading frame stop codon (sense, G G G G T A C C T C G A G C C T T C C T GCTTGTTCC); the sense primer also encoded sequential Kpn I and Xho I restriction endonuclease sites before PrP-related sequences. The PCR product was digested with Kpn I and Nar I, then ligated to phgPrP.NotI that had been cleaved with Kpn 1 and Nar I. (D) The fully assembled expression plasmid MoPrP.Xho.

B

~97 ~%%~~i~i~~

I

~ 68

Fig. 3. Demonstration of transgene-derived APP expression in brain. (A) Immunoblot analysis of total brain homogenate, using the monoclonal antibody 22Cll, which recognizes both human and mouse APP [1]. The brains of mice possessing transgene D N A (lanes 2, 3, 4, and 7; marked by ' -- ') contain 1.5-2 fold more APP than nontransgenic brains (lanes 1,5,6, and 8). Lane 2, from line Q2-2; lanes 3 and 4, from line El-2; lane 7 from line C3-3. (B) Immunoblot analysis of total brain homogenates, using the monoclonal antibody 6El0, which specifically recognizes human Aft [1,8,9].

D.R. Bor chelt et al. 'Genetic Analysis: Biomolecular Engineering 13 (1996) 159-163

Transgene +

+

+

+

+

1

2

3

4

5

6

--200

--97 --68

Fig. 4. Expression of transgene-derived APP. An immunoblot analysis of an equal volume of 10% (wt/v) homogenates of brain, heart, kidney, liver, and lung from transgenic mice, using the monoclonal antibody 6El0.

To further characterize transgene expression, we examined the distribution of transgene-derived polypeptides in mice harboring; human presenilin 1 (PS1) transgenes under the influence of the MoPrP.Xho vector [13]. Homogenates (10% (w/v)) of forebrain, heart, skeletal muscle, lung, liver, kidney, and spleen were prepared and equivalent volumes of each sample were analyzed by immunoblot with a PS1 antiserum (~PS1Loop) raised against epitopes in the 'loop' domain, which is near the C-terminus of PS1 [13]. Like APP, PS1 is ubiquitously expressed [14] and undergoes endoproteolytic processing. Tile 43 kDa precursor polypep-

tide of PS1 is cleaved to generate a N-terminal ( ~ 27 kDa) and C-terminal ( ~ 17 kDa) (loop-containing) derivatives [13]. The human and mouse C-terminal derivatives can be resolved by SDS-PAGE due to differences in apparent electrophoretic mobility [13]. Immunoblot analysis of tissue extracts from PS1 transgenic mice with antiserum to the C-terminal loop domain demonstrated the 17 kDa transgene-encoded human PS1 polypeptide in all adult tissues except spleen (Fig. 5A, filled arrow); endogenous mouse PS1 C-terminal fragment was detected in all tissues. Fulllength PS1 (43 kDa) was also detected in the brain, kidney, and heart (weakly) (Fig. 5A, open arrow). Because previous studies have demonstrated that the level of the 43 kDa polypeptide parallels levels of mRNA in the brain, whereas the level of the 17 kDa C-terminal derivative plateaus at relatively low levels of mRNA expression [13] we conclude that PS1 transgene expression is highest in the brain, kidney, and heart. The distribution of transgene expression in the brain was further investigated in PS1 transgenic mice, using in situ hybridization with a 33p-labeled antisense riboprobe generated from a human PS1 cDNA template (Fig. 6). Expression of transgene mRNA was sufficiently high to be visualized by standard autoradiography. The brains of two different lines of PS1 transgenic mice showed a similar distribution of transgene expression, with ,the densely packed CA fields of the hippocampus and the granular layer of the cerebellum showing the most robust hybridization. Selected nuclei (e.g. pons) as well as the cortex, thalamus and caudate also demonstrated robust hybridization. An examina-

br~ heart muscl(~ lung l i v e r kidney, spleen + +1 ++ + + 1 2 / 3 ÷4 ÷Is 5 110~ I 12 13 14115 16 17 18 19 20

A

,~_. %%

------

161

-68 -43 -29

~B

E~

-18 -14 -68 -43 -29 -18 -14

Fig. 5. Expression of transgene.-derived PS1. (A) An equal volume of 10% (wt/v) homogenates of the brain, heart, skeletal muscle, lung, liver, kidney, and spleen from PS1 transgenic mice expressing human PSl were analyzed by SDS-PAGE and immunoblot with PS1 'loop' antiserum as previously described [13]. Transgenic tissues marked by ( + ); open arrow, full-length 43 kDa human PSl polypeptide; filled arrow, 17 kDa human PS l C-terminal fragment; arrowhead, 16 kDa mouse PSl C-terminal fragment. (B) Identical volumes of extract were electrophoresed and gels were stained by Coomassie Blue to demonstrate similar protein loading.

D.R. Borchelt et al./ Genetic Analysis: Biomolecular Engineering I3 (I996) 159-163

162

pons

B

C ' f " W I,il;IL a , ~ l,#l,I t a I I I q~i I

[l lldl{llllU~t

C

Fig. 6. Distribution of PS1 transgene-derivedmRNA in brain. Brains, frozen in OTC freezingmedium, were sectioned and hybridized with a 33p-labeledriboprobe generatedfrom human PS1 cDNA. A, B, and C, autoradiographs of brains sections from previouslydescribed PS1 transgenic mice lines RS-1, B8-1 [13],and nontransgenicmice, respectively. tion of slides under higher magnification after emulsion development demonstrated transgene-encoded m R N A in astroglial cells as well as neurons (not shown). However, the relative level of expression in neurons as compared to glia is difficult to judge by in situ hybridization due to differences in cell size and volume. Based on these findings, we conclude that the MoPrP.Xho vector directs expression of transgenes in both neurons and astroglia of the CNS, with virtually all neurons demonstrating expression with the possible exception of cerebellar Purkinje cells [6].

3. Discussion Our investigations indicate that the MoPrP.Xho expression plasmid possesses sequences capable of directing the expression of foreign genes in the CNS of mice. Although PS1 transgene-derived polypeptides could be detected in a variety of other tissues, including skeletal muscle, lung, liver, and kidney, APP transgene-encoded polypeptides could only be detected in the brain and

heart. Although these data appear to be incompatible, the PS1 antiserum used in these studies is an extremely sensitive antibody, capable of detected picogram amounts of human PS1 [13] whereas the mAb 6El0, used to detected APP transgene-encoded polypeptide, requires nanogram levels of antigen to be present in a given sample (Borchelt, personal observation). Because PS1 is a relatively rare polypeptide, estimated to comprise 0.0003% of total protein in mouse brain (D. Borchelt and G. Thinakaran, personal observation), whereas APP is a relatively abundant protein ( ~ 0.05% of total protein in rat brain [15]), the demonstration of transgene-encoded PS1 polypeptide in tissues lacking transgene-encoded APP suggests that MoPrP.Xho vector is capable of driving low levels of transgene expression in a variety of tissues, with the highest expression occurring in the brain and heart. Although previous studies have demonstrated that endogenous PrP is approximately five times more abundant in the brain than in the heart, lung, intestine, kidney, testis, and muscle [16,17], the expression of recombinant APP in the brains and hearts of our transgenic mice was relatively similar. The basis for the apparently ectopic expression of the transgene in hearts of these mice is unclear, but may result from manipulations in the authentic genomic sequence or differences in the stability of APP and/or PrP m R N A and polypeptides in the heart. The MoPrP.Xho vector offers several features that make it a highly useful reagent for transgenic studies. First, our analyses of over 20 different lines of PS1 and APP transgenic mice revealed that the level of transgene expression in brain is relatively proportional to the number of integrated transgene copies, i.e. transgene expression is higher with higher numbers of transgene copies (data not shown). Second, because the entire open reading frame (ORF) of the prion protein gene is normally contained within a single exon, the replacement of the prion protein O R F with a recombinant cDNA ORF does not disrupt normal m R N A processing signals. Finally, the reduced size of the MoPrP.Xho vector, relative to the previously described SHaPrP.tet vector [2], allows for easy manipulation of the vector in a bacterial plasmid and, moreover, allows for the introduction of large cDNA, such as huntingtin ( ~ 10 kb cDNA). We believe that the MoPrP.Xho vector will be useful in creating transgenic models of autosomal diseases of the brain, and possibly heart, especially in those cases where overexpression of the transgene-encoded polypeptide may accelerate the onset of the phenotype as occurs in transgenic models of familial amyotrophic lateral sclerosis [18].

Acknowledgements The authors thank Deborah A. Swing for performing embryo microinjections; David Westaway, Carol

D.R. Borchelt et al./ Genetic" Analysis: Biomolecular Engineering 13 (1996) 159-163

Cooper, a n d Stanley Prusiner for m o u s e PrP D N A clones a n d sequence i n f o r m a t i o n ; D r G o p a l Thin a k a r a n for PS1 loop antiserum; D r A l l a n Levey for m o n o c l o n a i antibodies to h u m a n PS1; Drs Charles W e i s s m a n n , Philip W o n g , George C a r l s o n a n d K a r e n Hsiao for helpful discussions. We also t h a n k D u s t i n Englekin, Liesl Awalt, a n d L u b a R o m a n s t e v a for technical assistance. This work was supported by grants from the N a t i o n a l Institutes of Health in the form o f a n Alzheimer's Disease Research Center (DLP; 2 P50 Ag 05146) a n d a Leadership a n d Excellence in Alzheimer's Disease ( L E A D ) award ( D L P a n d D R B ; 1 R35 A G 07914), a n d grants from the Alzheimer's Disease a n d Related Disorders Association (DRB), The K a n t o n of Z u r i c h ( M F , CW), Schwiezerische N a t i o n a l f o n d s (CW), a n d the H u m a n F o n t i e r Science P r o g r a m (CW), a n d by the N a t i o n a l C a n c e r Institute, D H H S , u n d e r contract with A B L ( N C a n d N J). Requests for the vector should be addressed to D a v i d R. Borchelt.

References [1] Hsiao KK, Borchelt DR, Olson K, Johannsdottir R, Kitt C, Yunis W, Xu S, Eckman C, Younkin S, Price D, Iadecola C, Clark HB, Carlson G. Neuron 1995; 15: 1203-1218. [2] Scott MR, K6hler R, Foster D, Prusiner SB. Protein Sci 1992; I: 986 997. [3] Telling GC, Scott M, Hsiao KK, Foster D, Yang SL, Torchia M, Sidle KCL, Collinge J, DeArmond SJ, Prusiner SB. Proc Natl Acad Sci USA 1994; 91: 9936-9940. [4] Basler K, Oesch B, Scott M, Westaway D, W~ichli M, Groth DF, McKinley MP, Prusiner SB, Weissmann C. Cell 1986; 46: 417-428. [5] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, 1989. [6] Fischer M, Rfilicke T, Raeber A, Sailer A, Moser M, Oesch B,

163

Brandner S, Aguzzi A, Weissmann C. EMBO J 1995; 15: 12551264. [7] Mullan M, Crawford F, Axelman K, Houlden H, Lillius L, Winblad B, Lannfelt L. Nat Genet 1992; 1: 345-347. [8] Kim KS, Miller DL, Sapienze VJ, Chang CJ, Grundke-Iqbal I, Currie JR, Wisniewski HM. Neurosci Res Commun 1988; 2: 121-130. [9] Kim KS, Wen GY, Bancher C, Chen CMJ, Sapienza VJ, Hong H, Wisniewski HM. Neurosci Res Commun 1990; 7:113 122. [10] Kang J, Lemaire H-G, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H, Multhaup G, Beyreuther K, Mfiller-Hill B. Nature 1987; 325: 733-736. [11] Tanzi RE, Gusella JF, Watkins PC, Bruns GAP, St George-Hyslop P, Van Keuren ML, Patterson D, Pagan S, Kurnit DM, Neve RL: Amyloid/~ protein gene. Science 1987; 235:880 884. [12] Slunt HH, Thinakaran G, von Koch C, Lo ACY, Tanzi RE, Sisodia SS. J Biol Chem 1994; 269:2637 2644. [13] Thinakaran G, Borchelt DR, Lee MK, Slunt HH, Spitzer L, Kim G, Ratovitski T, Davenport F, Nordstedt C, Seeger M, Hardy J, Levey AI, Gandy SE, Jenkins N, Copeland N, Price DL, Sisodia SS. Neuron 1996; 17: 181-190. [14] Sherrington R, Rogaev El, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin J-F, Bruni AC, Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, Polinsky RJ, Wasco W, Da Silva HAR, Haines JL, PericakVance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St George-Hyslop PH. Nature 1995; 375:754 760. [15] Potempska A, Styles J, Mehta P, Kim KS, Miller DL. J Biol Chem 1991; 266:8464 8469. [16] Oesch B, Westaway D, W/ilchli M, McKinley MP, Kent SBH, Abersold R, Barry RA, Tempst P, Teplow DB, Hood LE, Prusiner SB, Weissmann C. Cell 1985; 40: 735-746. [17] Bendheim PE, Brown HR, Rudelli RD, Scala LJ, Goller NL, Wen GY, Kascsak RJ, Cashman NR, Bolton DC. Neurology 1992; 42: 149-156. [18] Wong PC, Pardo CA, Borchelt DR, Lee MK, Copeland NG, Jenkins NA, Sisodia SS, Cleveland DW, Price DL. Neuron 1995; 14:1105 1116. [19] Westaway D, Mirenda CA, Foster D, Zebarjadian Y, Scott M, Torchia M, Yang SL, Serban H, DeArmond SJ, Ebeling C, PrusinerSB, Carlson GA. Neuron 1991; 7: 59-68.