Analysis of gene expression using Simian Virus 40 vectors

ANALYTICAL BIOCHEMISTRY 135, 1 - 15 ( 1983)

REVIEW

Analysis of Gene Expression Using Simian Virus 40 Vectors

SURESH SUBRAMANI,*~’ AND PETER J. SOUTHERN?

*Department of Biology, B-022, Bonner Hall, University of California. San Diego, La Jolla, California 92093, and TDepartment of Immunology Scripps Clinic and Research Foundation, La Jolla, California 92037

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.

The introduction of exogenous DNA into eucaryotic cells has become an integral part of many current experimental protocols in- vestigating gene expression and regulation. This process requires efficient delivery of the DNA into the nucleus of the recipient cell and subsequent identification of cells that are expressing the foreign DNA. Depending on the DNA (intact gene, cDNA, or bacterial gene), it may be necessary to provide eucaryotic transcription signals to direct expression in recipient cells. The papova virus SV402 was developed initially as a eucaryotic-transducing vector using a lytic virus system (l-5). Sub- sequently, transforming (nonlytic) vectors were constructed with isolated segments of the SV40 genome (2-5). More recently, other vectors have also been developed with cloned genomes for bovine papilloma virus (6- lo), murine and avian retroviruses ( 1 l-16), ade- novirus ( 17), herpes virus ( 18), and vaccinia virus ( 19). The availability of increasing numbers of cloned cellular genes combined with

’ To whom correspondence and reprint requests should be addressed.

’ Abbreviation used: SV40, Simian virus 40.

techniques for in vitro mutagenesis (20) and the reintroduction of genes into appropriate cell types (21-25) (including fertilized mouse embryos that can develop fully into transgenic animals (26)) now permits meaningful studies of gene regulation and expression during dif- ferentiation and development.

Genome Organization of SV40

Genetic and biochemical information on the organization of the SV40 genome (re- viewed in (27)) has been confirmed or established by the nucleotide sequence of the viral genome (28,29). The design of different SV40 vector molecules has relied on the accurate mapping of genetic signals and the use of restriction endonucleases for the isolation of defined fragments from the SV40 genome. Spe- cific locations within the SV40 genome are identified either by map units (m.u.) or nucleotide numbers. The map units represent fractional genome lengths measured clockwise from the unique EcoRI restriction endonuclease recognition site (O/1.0 m.u.). The nucleotide numbers, derived from DNA-se- quencing studies, increase in a clockwise di-

1 OOO3-2697/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction m any form reserved.

2 SUBRAMANI AND SOUTHERN

rection from the center of a short palindromic sequence (map position 0.67) that constitutes part of the unique origin of DNA replication (ori). The SV40 genome contains 5243 base pairs (bp); therefore 0.1 map unit is equivalent to 524 bp (Fig. 1). SV40 nucleotide numbers in this paper are based on the SV numbering system described by Buchman et al. in Ap- pendix A of Ref. (27).

The expression of the SV40 genome is regulated in a temporal manner in infected cells. Shortly after infection, but before the onset of DNA replication, viral transcripts are syn-

thesized predominantly from one part of the viral genome (the early region). As the infeo tion progresses, viral DNA replication occurs and transcripts corresponding to the late region of the viral genome accumulate. The early mRNAs are transcribed from the E strand in a counterclockwise direction from the early promoter whereas the late mRNAs are transcribed from the other strand (the L strand) in the opposite direction (Fig. 1). The early and late-region transcripts represent complex families of mRNAs that differ with respect to transcription initiation and splice sites; and

PIG. 1. Physical and genetic map of SV40 DNA. The inner circle symbolizes the closed, circular DNA molecule; indicated within the circle are nucleotide numbers starting and ending at O/5243. The small arrows within the circle are sites for five restriction endonucleases that cleave SV40 DNA once. Map units in fractional-genome lengths beginning at O/l.0 are on the outside of the circle. Coding regions for viral proteins are shown as stippled arrows extending from the first nucleotide of the initiation codon to the last nucleotide of the termination codon. Each of the coding regions is embedded in an mRNA, the span of which is indicated by dotted or dashed 5’ ends and poly(A) 3’ ends. The wavy lines represent portions of the transcript that are spliced in forming the mature mRNAs.

SIMIAN VIRUS 40 VECTORS TO STUDY GENE EXPRESSION 3

there are unique polyadenylation sites for the early and late mRNAs.

Two classes of transcripts are synthesized from the SV40 early region. The shorter transcripts, which represent the major species, contain the coding sequence for SV40 large- T antigen (90-96K polypeptide) and lack 346 nucleotides (map units 0.60-0.54) that have been removed from the primary nuclear transcript by RNA splicing. The second class of early region mRNAs lacks 66 nucleotides (map position 0.54) from the primary early region transcript and is translated to small-t antigen (17-20K polypeptide). There are two types of large-T and small-t mRNAs (30-32); those with downstream start sites (located between nucleotides 523 1 and 5237) and others with 5’ ends lying further upstream (between nucleotides 30 and 35). The upstream start sites are used at the time of DNA replication and predominate late in infection ((30-32) Buchman and Berg, unpublished). The amino- terminal sequences of large-T and small-t antigen polypeptides are identical, but the translation phases of the two mRNAs are changed by RNA splicing, and hence the carboxyl terminal sequence of small-t antigen is unrelated to the corresponding region of large-T antigen. The translation-termination codon for small- t antigen lies within the intron of the large-T antigen mRNA.

Large-T antigen binds to the SV40 ori (33, 34) to initiate viral DNA replication during lytic infections and is also required for the initiation and maintenance of the transformed phenotype in nonpermissive cells. The expression of the SV40 early region is auto- regulated by large-T antigen so that the rate of early region transcription is reduced late in infection. The function of small-t antigen has not been clearly defined; there are viable SV40 mutants that synthesize either truncated forms or no detectable small-t antigen. This polypeptide, however, may contribute to the dis- ruption of the cytoskeleton in transformed cells.

Transcription of the late region produces 16 S and 19 S size classes of mRNA (about

1500 and 2300 bases, respectively). These mRNAs have variable 5’ nontranslated leader sequences (spanning the region 0.69-0.76 m.u.) that are spliced to the protein-coding sequences. The 16 S mRNA is the major late transcript and is translated to produce VP 1, the major structural component of the virion (VP1 , 40K polypeptide). The 16 S mRNA is spliced and sequences between map positions 0.765 and 0.94 are removed from the primary late-region transcript. The 19 S RNA is predominantly spliced between 0.76 and 0.765 m.u. or 0.73 and 0.765 m.u. but other minor forms, including apparently unspliced RNA, have been detected. The 19 S late mRNA is translated to produce the minor capsid proteins VP2 and VP3 (38.5K and 27K polypeptides, respectively). The amino acid sequence of VP3 is contained entirely within the carboxyl-terminal sequence of VP2. The VP2/VP3 coding region overlaps the VP1 coding region as translation occurs in two different phases in this segment of the SV40 genome. Recently, another late-region polypeptide, the agnoprotein, has been identified (35). This small polypeptide (8K) is encoded by an open-reading frame (0.72-0.76 m.u.) that re- sides within the 5’ leader sequences of the 16 S mRNA and certain species of 19 S mRNA.

Replication proceeds bidirectionally from the origin of DNA replication within the SV40 genome (map position 0.67). The point at which replication terminates (0.17 m.u.) ap- pears to be dispensable for replication. The viral DNA is encapsidated into virions as a supercoiled minichromosome and an opti- mum genome size is required for virion as- sembly. Genomes larger than SV40 are en- caps&ted very inefficiently whereas those that are significantly smaller than SV40 (4.5 kb or less) undergo sequence rearrangements, especially around ori, to increase the genome size.

SV40 Virus Vectors

SV40 vectors capable of being propagated as virions have been constructed with insertions into either the late or early region

4 SUBRAh4ANI AND SOUTHERN

(1,2,4,5,36). We have used the general no- genome. This imposes a maximum size limit menclature SVGTX-Y to describe these vec- (2-3 kb) for fragments that can be inserted tors; X is either an odd or an even number into SV40 virus vectors. In theory, larger fmg- according to the position of the insertion ments could be linked to an SV40 vector con- within either the late or early region, respec- taining just the origin of DNA replication but tively, and Y represents a simple abbreviation these recombinants could only be propagated to describe the inserted DNA. Thus SVGTS- in mixed infections with wild-type SV40. Un- /3G designates an insertion of the (rabbit) p- fortunately there is no selection for the hybrid globin cDNA between defined map positions genome and the wild-type SV40 rapidly out- in the late region of SV40 (Table 1). grows the hybrid genome.

Because there are no extensive dispensable regions within the SV40 genome, any insertion of foreign DNA must be accompanied by a corresponding deletion of viral DNA. Con- sequently, the virus vectors are defective and require a helper virus for propagation in permissive monkey cells. Complementation had been demonstrated between different pairs of SV40 virus mutants (37) and this approach was chosen for the propagation of various SV40 hybrid genomes with temperature-sensitive (ts) SV40 helper viruses at the nonpermissive temperature (4 1 “C) (38). If the hybrid viruses are to be propagated as part of the complementing pair then either the viral early or late region must be functional in the hybrid

Unlike bacteriophage X or plasmid vectors, none of the SV40 virus vectors exists as an independent viable entity; the vector fragments must be prepared from purified SV40 DNA with restriction endonucleases. Many of the virus vectors are based on recognition sites for the restriction endonuclease Hind111 (6 in total) present in the SV40 genome. Prepara- tion of the Hind111 vector fragments involves partial digestion with the enzyme to generate a random population of SV40 linear DNA molecules and purification of the linear molecules (from an agarose gel), followed by digestion with a second restriction enzyme (BamHI for late region vectors or &/I for early region vectors). The different vector fragments

TABLE 1

PROPERTIES OF SV40 VIRAL V~crom

Vector

Viral DNA replaced (m.u.)

Size of DNA replaced (kb)

Restriction sites at the ends of

vector Expression of foreign genes References

Late region SVGT 1 SVGT3 SVGTS SVGT7 SVGT9

Early region SVGTlO SVGT 12 SVGT14

0.725-o. 144 0.818-0.144 0.945-O. 144 0.859-0.144 0.986-O. 144

0.648-O. 189 0.324-o. 189 0,425-O. 189

2.2 HpuII-BumHI 1.7 HaeII-BumHI 1.0 HindIII-BumHI 1.5 HindIII-BumHI 0.8 HindIII-BumHI

2.4 HindIII-BclI 0.7 HindIII-BclI 1.2 HindIII-BclI

+ 70, 76, 77 +” 78 + 1, 43 +a 1, 43, 79 - 79

+ 41 - 41 - 41

a Transcripts initiating at the SV40 late promoter would contain AUG translation initiation codons 5’ to the translation-initiation codon of the inserted gene. Thus, it is likely that translation of the foreign gene occurs from internal AUG codons in the mRNAs. In view of the belief that translation of eucaryotic mRNAs initiates only from the first AUG in the mRNA (80), the mechanism of translation initiation from internal AUG codons remains unresolved.


are identified and recovered from agarose gels by reference to appropriate size markers.

Late-Region Virus Vectors

The vectors SVGTl, 3, 5, 7, and 9 (Table 1) permit the insertion of foreign DNA into the late region of the SV40 genome. The recombinant genomes generated from these vectors are propagated in monkey cells with a mutant containing a temperature-sensitive early region as helper (tsA58 is temperature sensitive for SV40 large-T antigen). All of the recombinant genomes retain a functional SV40 late promoter and the normal SV40 late region polyadenylation signal. The unique BamHl recognition site (map position 0.145) in the SV40 genome serves as a common end- point at the 3’ side of the inserted sequences, and the vectors therefore differ in the presence or absence of signals (5’ and 3’ intron junctions) that are required for normal splicing of SV40 late mRNAs.

SVGTl (Fig. 2) lacks the known SV40- splicing signals and this vector can be used to monitor the splicing and expression from inserted genomic DNA fragments. When the inserted DNA spans a complete gene (eg., rat insulin 1; Table 2), then several related transcripts may be observed; transcription initiation occurring at either the SV40 late pro-

moter or the inserted gene’s promoter and polyadenylation occurring at either the inserted gene’s polyadenylation site or at the late region site.

The SVGTS vector (Fig. 2) contains the splicing signals for both the 19 S and 16 S late mRNAs and the inserted sequence re- places the VP 1 coding sequence ( 1). With this arrangement, the inserted sequence can be transcribed very efficiently to produce a 16 S- type hybrid mRNA which can be translated to produce high levels of the foreign gene product. The inserted sequence is also present within the 19 S-type late mRNA but it is un- known if this mRNA is translated to produce the foreign gene product. The SVGTS vector has been used for efficient expression of bacterial genes and cDNAs in infected monkey cells (see Table 2).

The other vectors listed in Table 1, ie., SVGT3, SVGT7, and SVGT9, do not possess any unique features that make them more suitable than SVGTl or SVGTS. However, these vectors have been used and may be more appropriate under special circumstances.

Preparation of SV40 Late-Region Virus Vectors from Plasmid Intermediates

The late-region vector SVGTS has been used extensively to express bacterial genes or

AUG

FIG. 2. Structures of the viral vectors SVGTl and SVGTS. The dashes indicate segments removed from the vectors (see Table 1) and replaced by foreign DNA (Y) represented by the solid box. The symbol ori denotes the viral origin of DNA replication. In SVGT 1, the commonly used viral-splice donor and acceptor sites for late mRNAs are deleted, but the SV40 late-promoter and polyadenylation signals are intact. Hence, SVGTl is best suited for insertion of eucaryotic genomic sequences. The predicted structures of the 16 S and 19 Slate mRNAs transcribed from SVGTS-Y are also shown. The wavy lines represent introns removed from the RNAs by splicing. The VP2 and VP3 translation-initiation codons are upstream of the AUG codon in the foreign-gene segment.


TABLE 2

EXPRENON OF SOME FOREIGN GENES FROM SV40 VIRUS AND PLASMID VECTORS

Gene Virus vector

Rabbit &globin cDNA SVGTS, SVGT7, SVGTlO

Plasmid vector

psv2

Reference

1, 41

Rat insulin - 1 SVGT 1 70

Mouse /3”j globin SVGT3 16

Mouse cu-globin SVGTI 77

E. coli gpt pSVlGT5 psv2 psvzx-svgpt

5, 44

neo pSVlGT5 psv2 pSV2-X-SVneo

45

Mouse dhfr EDNA SVGTS SVGT7

pSVlGT5, pSV2 psvz-x-svdhrf

43,13

Human fibroblast interferon

psvz 46

Functionally rearranged immunogfobulin K-chain gene

pSV2gpt-K 60,81

E. coli cat psv2 3, 71

E. coli palactokinase osv2 3, 72

cDNAs in infected monkey cells (see Table 2). In order to simplify the construction of new recombinant genomes, the SVGTS vector has been joined to a segment of pBR322 to generate the plasmid pSVlGT5 (Fig. 3). The pSV 1 plasmids contain a small duplication of SV40 sequence (0.32-0.14 m.u.) in an arrangement that allows the SV40 genome to be inserted into pBR322 while preserving intact the viral early and late regions.

Derivatives of pSV1 can be propagated in Escherichiu cdi recA strains (eg., HB 10 1) and the SV40 hybrid genomes can be retrieved from the plasmids either in vitro or in vivo. The duplicated SV40 sequence in pSV1 includes the restriction enzyme recognition sites for PstI and MI, and either enzyme could be used to excise the SVGTX viral genome, which is ligated at low DNA concentrations in vitro to promote intramolecular circulari- zation and then introduced into CVlP mon-

key cells with tsA58 helper DNA. The majority of virus plaques that appear contain exclu- sively the predicted recombinant genome plus tsA58 helper. The PstI and MI restriction sites are located within the large-T antigen coding sequence and the sites must be reconstructed precisely in order to retain functional

FIG. 3. Structure of pSV1GTS-gpt. The solid black segment represents the pBR322 sequence, the white areas indicate SV40 sequences, and the hatched portion &notes the foreign DNA (5,48). The viral early and late regions are shown and the numbers represent SV40 map units.


large-T antigen. This method of releasing the hybrid genome may not be convenient if the inserted DNA contains multiple Z’stI or BclI sites. Note also that plasmid DNA prepared in HBlO 1 and most other E. coli strains is resistant to digestion with Bcfl because of methylation within the enzyme-recognition sequence by the bacterial adenine methylase. A mutant E. coli strain (dam) lacks this methylase (39) and may be used to prepare plasmid DNAs susceptible to digestion by &/I.

The recombinant viral genome can also be generated from pSV 1 although less efficiently in vivo by homologous recombination (40) in the duplicated sequences. A mixture of the pSVlGT5 hybrid DNA and tsA58 DNA is introduced into cells at the nonpermissive temperature (41°C). Since pSV1 is too large to be encapsidated into virions, plaque formation with the helper virus is dependent upon precise recombination to reconstruct a functional large-T antigen gene.

Early Region Virus Vectors

Foreign DNA sequences have also been inserted at various positions in the SV40 early region (SVGTlO, 12, and 14, Table 1) and the recombinants propagated by complementation with tsB201 helper virus ((41), ts B201 produces a temperature-sensitive VPl). Al- ternatively, early region recombinants may be propagated without a helper virus in the COS cell line which synthesizes SV40 large-T antigen constitutively (42).

In SVGTlO+G the &globin cDNA sequence is inserted at the Hind111 site (map position 0.645) close to the 5’ end of the early region. This arrangement removes the SV40 large-T antigen translation-initiation codon and ensures that the @globin initiator AUG is the first initiation signal for translation in the hybrid transcripts. A segment of vector DNA downstream from the @globin cDNA includes the SV40 small-t antigen-intervening sequence to permit splicing of the hybrid transcripts. The amount of &globin synthesized in SVGTlO-@G-infected cells is about one

hundred times lower than the amount of @- globin produced in comparable infections with SVGTS-PG.

Advantages and Disadvantages of SV40 Virus Vectors

The SV40 virus vectors provide an efficient method to study foreign gene expression in virus-infected cells. At high multiplicities of infection ( 1 O-50 pfu/cell), essentially the entire cell population is infected. As a consequence of DNA replication, the recombinant genome is extensively amplified and transcripts that include the inserted sequences accumulate to high levels. In many cases these hybrid transcripts produce large amounts of the predicted foreign-gene products. The late-region vectors SVGT 1, 3, 5, and 7 are particularly suited for the efficient synthesis of foreign-gene products.

The complementation system for the propagation of recombinant viral genomes operates very effectively. The ratio of recombinant to helper genomes is normally about 1: 1, and recombinants that contain a duplication of the viral origin of DNA replication are propagated preferentially with respect to the helper. There is no evidence for appreciable recombination between mutant and helper genomes.

Some limitations of the SV40 virus vector systems preclude certain types of experiments. There is a strict limit on the size of fragments that can be inserted into the SV40 genome; recombinant genomes larger than wild-type SV40 are not encapsidated efficiently and accumulate deletions. The recombinant viruses can only be propagated in permissive monkey cells thereby excluding many specialized cell types as hosts. Permissive cells are also killed following virus infection and this precludes any long-term study of gene expression.

The preparation of homogeneous virus stocks can be time-consuming and expensive in terms of tissue-culture supplies. Often, independent plaques from DNA transfections contain a mixture of the helper, desired recombinant, and contaminating rearranged genomes. Virus plaque purification is neces-


sary to clone the specific recombinant genomes. The pSV 1GT5 plasmid vector represents a partial solution to this problem because, once the correct structure has been obtained in E. coli, there is a very high prob- ability of obtaining only the desired recombinant viral genome in transfected monkey cells. Certain genes or nucleotide sequences (eg., herpes simplex virus thymidine kinase, Fromm and Berg, unpublished, and E. coli xanthine-guanine phosphoribosyl transferase, (5)) are difficult to propagate in SV40 late- region vectors. These recombinants rapidly undergo deletions and/or sequence rearrangements. It is not clear whether this instability reflects the detrimental consequences of over- expression of the foreign-gene product or the existence of sequence elements in the inserted DNAs that promote deletions. At present, it is not possible to predict whether any given DNA fragment will be maintained stably by SV40 virus vectors, but rearrangements are the exception rather than the rule.

Several examples of aberrant splicing have been observed with the SV40 late-region vectors. Occasionally, a sequence within the inserted segment fortuitously provides a splicing signal. The consequent aberrant splicing can confuse the analysis of transcripts produced by the recombinant genomes and, where such cryptic splice sites reside within protein-coding sequences, the production of protein can be reduced.

The SV40 hybrid plasmid vectors were developed to overcome many of the disadvantages of virus vectors.

SV40 Hybrid Plasmid Vectors

These vectors contain pBR322 sequences that allow propagation of recombinants in E. coli and, in several cases, include DNA segments containing the SV40 or polyoma early regions which allow at least transient replication of these plasmids in permissive mammalian cells. The vectors contain a eucaryotic- transcription unit that directs the expression of inserted cDNA segments, eg., rabbit /3-glo-

bin (Howard and Berg, unpublished) and mouse dihydrofolate reductase (43), or bat terial genes, eg., the E. coli gpt gene coding for xanthine-guanine phosphoribosyl transferase ($44) or the neo gene coding for an aminoglycoside phosphotransferase (45). Ad- ditional DNA segments coding for other genes of interest (eg., human fibroblast interferon, (46)) may be inserted into the vectors at convenient restriction-endonuclease sites located in nonessential regions. These plasmid vectors surmount many of the limitations of the SV40 viral vectors, but the viral and plasmid systems are not mutually exclusive and the appropriate choice for a particular experiment should be judged carefully.

Plasmids of the pSV series and their derivatives have been constructed by combining restriction fragments in vitro and propagating the recombinant genomes in E. coli. This fea- ture of cloning in bacteria rather than in mammalian cells offers a rapid, convenient, and relatively inexpensive means for the preparation and purification of recombinant genomes. Moreover, since the length of the DNA insert is not limited by the SV40-packaging constraint, considerably larger DNA fragments may be introduced into cells.

The plasmid DNAs are introduced into mammalian cells by calcium phosphate pre- cipitation (22,23), DEAE-Dextran precipi- tation (47), microinjection (2 1,26), liposome fusion (24), or protoplast fusion (25). The ef- ficiency of DNA uptake varies with the method and cell line used.

During the initial 1-4 days following the introduction of DNA into mammalian cells (DNA transfection), transient expression of RNA and protein from the input DNA can be detected (48). Only a fraction of the cells (0.1-1.0s) that initially express the plasmid genes continue to do so for an extended period. These cells are stably transformed by the pSV plasmids by virtue of the integration of vector DNA into the host genome. Thus, only 1 in about 104- lo5 of the transfected cell population becomes stably transformed, and these rare transformants can only be identified if


expression of the foreign gene from the pSV vector causes a phenotypic change. The initial experiments of this type were performed with the herpes thymidine kinase (tk) gene using the tk-deficient recipient cell lines (49-50). Subsequently, other selectable markers like aprt, hgprt, and dhfr were used but these are restricted to auxotrophic cell lines that have the appropriate deficiency. Dominant genetic markers which produce a selectable alteration in the phenotype of normal cells permit transformation of any host cell. At present, three dominant selectable markers-gpt (5,44), neo (455 1), and a methotrexate-resistant dhfr cDNA (52)-are available, of which the for- mer two have been used extensively with the pSV vectors for cell transformation.

The expression of the bacterial gpt gene permits the utilization of xanthine as a sub- strate for the purine-salvage pathway and selection conditions have been established in which gpt functions as a dominant marker (5,44). The expression of the bacterial neo gene protects mammalian cells from the cytotoxic

action of the aminoglycoside drug, G418 (45,5 1).

Any other gene which does not by itself confer a direct selection may be introduced into cells by linkage with either the gpt or neo genes, and the resulting transformants can then be examined for the expression of the nonselected gene (see Ref. (46)).

Properties of pSV2

The prototype plasmid vector, pSV2 (Fig. 4) contains a segment of pBR322 DNA that includes the plasmid origin of DNA replication and the P-lactamase gene to permit propagation and selection (ampicillin resistance) in E. cob. The SV40 sequences in pSV2 are derived from the viral early region and constitute a eucaryotic transcription unit-promoter, intervening sequence, and polyadenylation site. Foreign DNA (cDNA or genomic sequence) is inserted into the pSV2 vector between the promoter and intervening sequence. In this arrangement, translation-initiation co-

AmpR

pBR322 ori

FIG. 4. The SV40 plasmid vector pSV2. The prototype pSV2 (constructed by B. Howard, Howard and Berg, unpublished) is shown on the left. The 2296-bp segment from the EcoR 1 -pVuII site (counterclockwise) contains the @-lactamase gene and the origin of replication from pBR322. The 342-bp PvuII-Hind111 fragment (nucleotide 270-5 172 in SV40) contains the SV40 ori and the promoters for early (counterclockwise) and late (clockwise) transcription. Foreign DNA that is to be expressed from the SV40 early promoter may be inserted into the space between the Hind111 and BgflI sites. The DNA from the Bg/II-EcoRI site contains two SV40 DNA fragments ligated together-a 610-bp Mb01 fragment (nucleotide 4710-4100 in SV40) linked to a 988-bp &/I-EcoRI fragment (nucleotide 2770-1782 in SV40). The BgfII site is present by virtue of the fact that cleavage at the Mb01 site at bp 4710 in SV40 happens to generate half a BgflI site. The 6 1 0-bp SV40 fragment provides the intron from the small-t antigen gene while the BclI-EcoRI SV40 fragment supplies a polyadenylation signal. In pSV2-gpt, a 1062-bp HindIII-BumH 1 fragment containing the E. coli gpt gene is inserted between the Hi&III and BgfiI ends of the pSV2 vector on the left. Consequently, there is no BgfiI site at the end of the gpt gene in pSV2-gpt. pSV2 vectors with dhfr, gpt, or neo markers all contain unique BamHI and EcoRl sites into which other transcription units can be inserted. The DNA between the BumHI and EcoRI sites can be removed without affecting expression from the SV40 promoter.


dons within the inserted sequences can be rec- ognized correctly and the hybrid mRNA can be translated to give the expected protein products.

The original pSV2 plasmid contained a rabbit @globin cDNA sequence between the Hind111 and BgfiI sites (pSV2+3G). The vector fragment is prepared by complete digestion of pSV2+3G with Hind111 and Bg!II to remove the @-globin fragment. Any coding sequence (Y) may be inserted between these sites to generate a recombinant of the structure pSV2- Y. The inserted sequence must have a Hi&III- cohesive site at the 5’ end and a 5’-GATC-3’- cohesive site at its 3’end. After in vitro ligation of pSV2 with the insert segment, the pSV2- Y recombinants are propagated in E. coli prior to their introduction into mammalian cells. While the HindIII site is maintained in pSV2- Y recombinants, the BgflI site is reconstructed only if BglII is used to generate the cohesive end at the 3’ end of the inserted fragment.

Although pSV2 contains the SV40 ori, it cannot replicate in monkey cells because of the absence of SV40 large-T antigen. Stable propagation of pSV2 plasmids in mammalian cells is therefore dependent upon integration into chromosomal DNA. Recently, autono- mous pSV2 piasmid DNA replication has been demonstrated in COS cells (Smith, Southern, and Berg, unpublished; (5 3)) which synthesize large-T antigen constitutively.

The expression of coding sequences in pSV2-Y depends upon the signals provided by the DNA segments. Since many mammalian cells including mouse, rat, hamster, and human are stably transformed by SV40 (27), these cells should support the constitutive expression of integrated pSVZY sequences. Moreover, in the absence of large-T antigen, repression of the SV40 early promoter cannot occur.

A variety of other SV40 shuttle vectors capable of being propagated in both pro- and eucaryotic cells have also been constructed in a number of different laboratories. Many of these lack selectable marker genes and have been used primarily to study transient expres-

sion of cloned genes (54-56). For example, pBR322 derivatives containing only the SV40 origin of DNA replication are also capable of replicating extrachromosomally in COS cells (57). It has also been reported that pBR322 contains certain “poison” sequences that in- hibit plasmid replication in eucaryotic cells (58). Consequently, SV40-pBR322 hybrid plasmids lacking these poison sequences have also been used (54,58).

Properties of Cells Transformed with pSV Vectors

The pSV2 vector containing either dhl? (43) gpt (5,44), or neo (45) marker genes transforms cells at a frequency of 10-4-10-5. This frequency, defined as the number of transformants obtained per 10’ cells using 10 pg of the vector DNA (with no added carrier DNA), is observed with the modified calcium phosphate-transfection protocol (23). Recently, transformation frequencies as high as 5 X lo-* have been achieved with pSV vectors (3). At present, the dhfr cDNA plasmids can only be used to transform dhfr-deficient cell lines, whereas the gpt and neo markers can be used with a wide variety of normal cells (Table 3). The majority of transformants that have been analyzed contain l-5 copies of the plasmid DNA integrated independently into the chromosomal DNA. Multiple insertions and du- plications or rearrangements of plasmid sequences after integration are comparatively rare. Transformants of the COS cells, in which the pSV2 plasmids replicate efficiently, do not contain integrated DNA, but have extrachro- mosomal DNA instead (Smith, Southern, and Berg, unpublished, (5 3)).

The integrated plasmid sequences are retained stably even when transformed cells are maintained nonselectively. For example, two pSV2-neo-transformed cell lines were pas- saged for over 100 cell generations in normal medium and there was no indication of either loss or rearrangement of the pSV2-neo sequences (45). It has also been possible to grow the cells nonselectively for 10 generations after


TABLE 3

SOME CELL LINES TRANSFORMED WITH THE gpt AND neo DOMINANT SELECTABLE MARKERS

Cell line Type Marker Reference

3T3 3T6 PCC4 F9 MEL L Ltk- cv-I CVl-P TC7 cos HeLa LNSV

K562 XP6Be

CHO-dhfr- SlA-2 Y3-Ag1.2.3 27-44 BW5147

Mouse Mouse Mouse Mouse Mouse erythroleukemia Mouse Mouse Monkey Monkey Monkey Monkey Human SV40 transformed Human (HPRT-) Human SV40 transformed human Xeroderma pigmentosum line Chinese hamster ovary Mouse lymphoid (HPRT-) Rat myeloma (HPRT-) Mouse hybridoma (HPRT-) AKR thymoma (HPRT-, Oua’)

neo 45 neo, gpt 5. 44, 45 neo 45 neo 45 neo, gpt 45 neo, gpt 45 neo, gpt 45 neo, gpt 5, 44, 45 neo, gpt 5, 44, 45 neo, at 5, 44, 45 neo, gpt 53, 74 neo, fm 45 neo, iwt 5, 44, 45

neo 45 gpt 82

gpt 73 gpt 81 gpt 60 gpt 60 gpt 60

the introduction of pSV-neo plasmids and then to apply the selection. Under these conditions, transformed cells were recovered at 20% the frequency of that found in the usual transformation/selection; this suggests that plasmid sequences are maintained stably after integration into chromosomal DNA even in the absence of selective pressure (45).

Derivatives of pSV2 with Altered or Additional Transcription Units pSV2-X-svgpt

The eucaryotic transcription unit in pSV2 contains all the signals believed to be involved in gene expression in mammalian cells. Therefore, by monitoring the expression of the marker gene, it is possible to determine the effects of removing or replacing the SV40- derived transcription signals. One plasmid, pSV0, lacks the SV40 early promoter and origin of replication. This change eliminates expression of marker genes and reduces the transformation frequency to about 1 in lo7

There are occasions when it is desirable to transfect mammalian cells with cDNAs or coding sequences which do not confer any selection. If the nonselected sequence (X) is to be expressed from the pSV2 transcription unit, a pSV2-X derivative is first constructed and then a second transcription unit (SVgpt, SVdhfr, or SVneo) is inserted into pSV2-X at the unique BamHl site. The resulting recombinant, pSV2-X-SVgpt contains tan- demly arranged transcription units, each of

cells. Since expression of the marker is dependent on the presence of a functional eucaryotic promoter, pSVO-gpt can be employed to test DNA segments for promoter activity (59). In a similar manner, the importance of RNA splicing or polyadenylation signals for the expression of marker genes can be analyzed by removing, altering, or replacing these signals.


which can potentially be expressed from identical genetic signals (Fig. 5). Cells selected for the acquisition and maintenance of a functional gpt (or dhfr or neo) transcription unit can then be examined for the presence and expression of the nonselectable coding sequence X.

Plasmids of the type pSV2-dhfr-SVgpt and pSV2-neo-SVgpt have been constructed and transformants obtained by selecting for one or other marker. Greater than 50% of these transformants, selected initially for either marker, also express the nonselected marker. This approach is also applicable for genomic DNA sequences which are inserted directly into pSV2 marker plasmids and introduced into mammalian cells by selection for expression of the marker gene, as has been demonstrated for the human fibroblast interferon (46) and K light chain (60) genes.

Detection Systems for Foreign DNA and Foreign Gene Expression Southern-blotting experiments (6 1) with

whole cell DNA preparations and either all or part of the transforming DNA as a hybridization probe show that the foreign DNA usually integrates at random locations into chromosomal DNA of transformed cells (49). Reconstruction experiments can be used to provide an accurate estimate of the copy number of integrated sequences and system- atic restriction-endonuclease mapping reveals

SV40 ori gpt

FIG. 5. Structure of pSV2-X-SV gpt. The solid area represents pBR322, the stippled portion denotes SV40 sequences, and the clear and crosshatched segments show any foreign gene X and the gpt gene, respectively. This vector contains two complete and independent transcrip- tional units, one expressing X and the other gpt.

information relating to the junctions between chromosomal and transforming DNA sequences (62). The majority of the transformants generated with the pSV vectors contain l-5 integrated plasmid genomes; occasional clones contain very high copy numbers and may have multimeric arrays of the plasmid genome. As a rapid, preliminary screen, whole- cell DNA samples can be examined by the DNA dot-blot procedure (63); this is especially useful in screening transformants for nonselected DNA sequences and for approximate estimations of integrated-sequence copy number.

In SV40 virus-infected cells, the viral DNA can be purified away from chromosomal DNA using the Hirt-extraction procedure (64). As mentioned earlier, in certain cells transformed with the selectable markers, the vector may exist as autonomously replicating DNA. The Hit-t method is also applicable for the re- covery of free (unintegrated) plasmid DNA in transfection/transformation experiments (53,57,58).

Transcription of foreign genes can be as- sessed by analyzing the cellular RNA (total, cytoplasmic, and nuclear separately, or polyA+/polyA- fractions as appropriate) either by “Northern” transfer and hybridization (65) or by S 1 nuclease protection experiments (66). Often the level of transcription does not cor- relate with the gene-copy number suggesting that the integrated sequences may be transcribed to varying degrees. Again, the dot- blot procedure may be applicable to screen clones rapidly for the presence of particular RNA species. This has been demonstrated successfully with inducible foreign-gene expression but the technique may not be sufficiently sensitive to detect low level transcription. The laboratory manual of Maniatis et al. (67) is an excellent source for many of the experimental protocols referred to in this sec- tion.

Synthesis of foreign proteins in transformed cells has been examined with immunological reagents and/or enzymatic assays. If the level of protein is sufficiently high, then cytoplasmic


or surface immunofluorescence with antibody conjugated to fluorescein or rhodamine prob- ably represents the most convenient detection technique. More commonly, transformed cells are cultured in the presence of radioactively labeled amino acids and then the total soluble protein is analyzed by immunoprecipitation (either protein A selection of immune com- plexes (68) or by the “Western” blotting procedure, (69)). Occasionally, foreign proteins are secreted. For example, insulin is secreted as a preprotein from fibroblast cells and normally represents the only labeled protein in the culture medium (36,70).

Several of the marker genes for transformation (tk (49), gpt (5), dhfr (43), and neo (45)) code for enzymatically active polypeptides which can be conveniently assayed. Re- cently, the chloramphenicol acetyltransferase (cat) gene (71) from Tn9 has been used to examine promoter function in transformed eucaryotic cells. Although the cat gene does not confer any selection in eucaryotic cells, the enzyme activity can be quantitated in a rapid and highly reproducible assay (7 1). The E. coli galactokinase (galK) gene has also been expressed from pSV2 (72).

Advantages and Disadvantages of SV40 Hybrid Plasmid Vectors

The plasmid vectors provide an opportunity to study gene expression and regulation in a variety of mammalian cells. The plasmids do not replicate autonomously (except in COS cells) and propagation is dependent on stable integration into chromosomal DNA. Because stable integration is rare, it is necessary to use selectable markers to recognize cells which ac- quire and express the foreign DNA. The availability of dominant selectable markers (gpt and neo) permits the use of virtually any cell type as a recipient in DNA-transfer experiments (Table 3).

Once integrated, the plasmid sequences are maintained indefinitely even when the cells are passed nonselectively. The altered phenotype is also retained without selection, in-

dicating that the marker genes are expressed constitutively in transformed cells. However, the majority of independent transformants only contain a few copies of integrated DNA and the level of expression of the marker gene may be low. Methods for amplification (73) of plasmid sequences and/or integration of large aggregates of plasmid DNA are being studied in order to increase the plasmid-copy number and the level of expression of foreign genes in transformed cells.

The plasmid vectors can also be used to monitor gene expression in short-term transfection experiments. This approach is attrac- tive because information on the levels of expression can be obtained rapidly (typically, gene expression is monitored l-3 days after transfection). Unfortunately, DNA transfection is not as efficient as SV40 virus infection and the level of gene expression is reduced 1 O- to 20-fold.

The plasmid vectors can be manipulated rapidly and easily in bacterial cells. Numerous derivative structures have been constructed by removal and/or replacement of SV40 tran- scriptional signals, and a comparison of these related genomes has contributed to an understanding of the parameters involved in eucaryotic gene expression. At present, there are a few methods to retrieve the integrated plasmid sequences in bacteria. It is possible to recover plasmid sequences by transformation of E. coli following fusion of transformants containing pSV DNAs with COS cells (74). The autonomously replicating pSV2 plasmids present in transformants of COS cells have also been recovered efficiently in E. coli (53), and it is anticipated that any autonomously replicating plasmid could be recovered from mammalian cells in a similar manner (see Refs. (6,9)).

The SV40 viral and hybrid plasmid vectors permit functional assays for the different reg- ulatory and coding sequences that are now being identified and isolated. The widespread use of these and other vectors (see Ref. (75) for the proceedings of a recent meeting on the subject) combined with the elegant methods


for in vitro mutagenesis should provide the basis for a better understanding of gene expression and regulation in mammalian cells.

ACKNOWLEDGMENTS

We would like to thank Paul Berg for his encouragement and support and also for providing the exciting environ- ment at Stanford where much of this work was done. We would also like to acknowledge Paul Berg, Richard Mul- ligan, Bruce Howard, and Andy Buchman for their valu- able contributions to the development of the vectors and strategies for their use. S.S. is being supported by Grants 5 ROl GM31253-02 from NIH and the Searle Scholars Program. P.J.S. was supported at Stanford by a postdoc- toral fellow ship from NATO and the British Science Re- search Council.

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Analysis of gene expression using Simian Virus 40 vectors

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