Cell, Vol. 37, 1053-1062, July 1984, Copyright 0 1984 by MIT 0092.8674/84/071053-i 0 $02.00/O Construction and Applications of a Highly Transmissible Murine Shuttle Vector

Constance L. Cepko, Bryan E. Roberts,* and sion of cloned in which the use of specialized Richard C. Mulligan recipients or whole is often crucial. In addition, a Department of Biology and Center for Cancer Research highly efficient transfer system is also important, both Massachusetts Institute of Technology for introducing genes into a minor component of a heter- Cambridge, Massachusetts 02139 ogenous population (e.g., hematopoietic stem cells in Whitehead Institute for Biomedical Research bone marrow tissue) and for studies in which gene transfer Cambridge, Massachusetts 02139 is used as a basis for gene isolation (Perucho et al., 1980; Okayama and Berg, 1983). Finally, it is often advantageous to be able to rapidly recover sequences introduced into Summary mammalian cells as molecular clones, both to analyze the structure of the transduced sequences and to recover the We develop a murine retrovirus shuttle vector sys- chromosomal DNA sequences flanking the site of insertion. tem for the efficient introduction of selectable and Therefore, a system that permits the “shuttling” of vector nonselectable DNA sequences into mammalian cells recombinants between and bacterial cells (DiMaio and recovery of the inserted sequences as molecu- et al., 1982) is particularly useful. lar clones. Three protocols allow rapid recovery of Here we describe the generation and characterization vector DNA sequences from mammalian cells. Two of a murine retrovirus vector system that meets these of the methods rely on SV40 T-antigen-mediated requirements. The system allows for the efficient conver- replication of the vector sequences and yield thou- sion of vector DNA into highly transmissible capable sands of bacterial transformants per 5 x lo6 mam- of infecting many cell types both in vitro and in vivo. The malian ceils. The majority of recovered by vectors make use of the strong retrovirus transcriptional all three protocols exhibited the proper structure and signals to promote the expression of both dominant se- were as active as the parental vector in the gener- lectable markers and nonselectable cDNA sequences. The ation of transmissible retrovirus upon vectors also allow for the rapid recovery of integrated transfection of mammalian cells. One of the rescue proviral sequences from mammalian cells as bacterial methods, which relies on “onion skin” replication plasmids. This latter feature has been successfully applied and excision of an integrated from the host towards the isolation of cellular sequences flanking provi- , enables facile recovery of the chro- rus integration sites and the isolation of cDNA sequences mosomal site of proviral integration. The system was generated directly by passage of genomic DNA se- also used to generate, and then efficiently recover, quences as part of a recombinant retrovirus . a cDNA version of a genomic insert from the ade- novirus ElA region. Results

Introduction Vector Design and Transmissibility The pZIP-Neo SV(X)I vector (hereafter termed SVX, shown The development of mammalian transducing vectors has in Figure I), consists of a Moloney murine leukemia virus been motivated by a variety of interests. Most of these (M-MuLV) transcriptional unit derived from an integrated interests relate to the prospect of reintroducing cloned M-MuLV provirus (Hoffman et al., 1982) and pBR322 genes into particular cell types in order to study either the sequences necessary for the propagation of the vector functional domains of proteins (Gething and Sambrook, DNA in E. coli. The specific M-MuLV sequences retained 1981; Rose and Bergmann, 1982) the interactions among in the vector include the long terminal repeats (LTRs) different polypeptides (Land et al., 1983; Ruley, 1983) or necessary for the initiation of viral and the the regulatory elements that affect the expression of par- polyadenylation of viral transcripts, as well as for integra- ticular genes (McKnight and Kingsbury, 1982; Chao et al., tion; sequences necessary for the reverse transcription of 1983; Green et al., 1983). In addition, with the recognition the viral genome; sequences for the encapsidation of viral that a number of biologically and commerically important RNA (Mann et al., 1983); and 5’ and 3’ splicing signals proteins required postranslational modifications unique to involved in the generation of the subgenomic viral env animal cells, there has been considerable interest in the RNA. In place of the retroviral sequences encoding the use of mammalian vectors to simply overproduce gene gag-pal and env polypeptides, two unique restriction en- products in animal cells. donuclease cleavage sites (Barn HI and Xho I) were in- To meet such varied applications, a mammalian gene serted, to permit the expression of sequences introduced transfer system must fulfill a number of requirements. First, into the vector from either the full-length or spliced retroviral the system should permit the introduction of genes into a transcripts (Weiss et al., 1982). DNA sequences derived wide range of recipients, both in vitro and in vivo. This is from the transposon Tn5, which encode G418 resistance particularly important for studies of the regulated expres- in mammalian cells (Davies and Jimenez, 1980; Colbere- Garapin et al., 1981; Southern and Berg, 1982) and kana-

* Permanent address: Department of Biological Chemistry, Harvard Medical mycin resistance in E. coli (Jorgensen et al., 1979) as well School, Boston, Massachusetts 02115. as sequences encoding the SV40 and pBR322 origins of Cell 1054

pZIP-NeoSV(X)I DNA from a variety of G418 resistant colonies confirmed [ cDNA -._ _,* these findings (data not shown). The transcriptional activity -._, Born1 of the integrated SVX genome was demonstrated in sev- -1 eral ways. Northern analysis of the RNAs produced in SVX transformants indicated that two viral RNAs, corresponding in size to the expected genomic and spliced transcripts, were produced in equal abundance (Ft. Cone, unpublished pZIP-Neo SV(B) I data). Furthermore, superinfection of, SVX transformants with wild-type M-MuLV resulted in cell lines that produced titers of SVX virus of up to 10’ cfu/ml.

Figure 1. Structure of the ZlPNeo Shuttle Vectors Recovery of Integrated Proviral Genomes as The pZlP provirus clone of Hoffmann et al. (1982) was used as the source Molecular Clones of all M-MuLV sequences. From the 5’ LTR to the Barn HI site was formally Three protocols were developed for the facile recovery of the O-1 .O kb fragment of pZlP (the Pst I site at nucleotide 566 [Shinnick et integrated SVX as bacterial plasmids. al., 19811 was converted to a Barn HI site by the addition of a linker). The 3’ SS fragment extends from the M-MuLV Bgl II site at nucleotide 5409 to Nonamplified Rescue the Xba I site at nucleotide 5768, and encodes a 3’ splice acceptor This method relies upon the generation of free circular sequence utilized in the formation of the subgenomic env RNA. The neo proviral forms prior to retrovirus integration (Weiss et al., fragment was originally the 1.4 kb Hind Ill-Sal I fragment from Tn5 (Jorgen- 1982). NIH3T3 cells were cocultivated with the SVX 2B sen et al., 1979; Southern and Berg, 1982). Sequences labeled SVori were derived from pSVOD (Mellon et al., 1981) and extend from SV40 nucleotide producer cell line for 20 hr, and Hirt DNA (Hi& 1967) 160 to nucleotide 5171 (Tooze, 1981). The pBRori sequences extend from containing putative closed circular proviral molecules was nucleotide 2521 to nucleotide 3102 (Sutcliffe, 1978) (the orientation of these isolated and used to transform E. coli. Transformants were sequences has not been established). The 3’ end of the vector was taken selected on media containing kanamycin sulfate and quick from pZIP, from the Hpa I site at nucleotide 7197 to the 3’ end of the pZlP plasmid DNA preps were prepared from individual colonies provirus. The restriction sites that were created during the construction are as shown. For reference of pZlP sites, the standard M-MuLV map can be to characterize the structure of the rescued viral genomes. consulted (Shinnick et al., 1981; Weiss et al., 1982). As summarized in Table 1, a majority of the plasmids exhibited the proper structure by restriction endonuclease replication, have been introduced into the Xho I site. These analysis. Furthermore, upon retransfection of the rescued sequences allow for the selection of mammalian cells genomes into +2 cells, all of the plasmids yielded at least harboring the SVX provirus and the rapid recovery of free as much SVX virus as the parental plasmid (Table 1). or integrated proviral genomes as bacterial clones (see Amplified Plasmid Rescue Following infection of below). In a related vector, SVB (Figure l), the Tn5, SV40, Cos Cells and pBR322 sequences were inserted into the Barn HI Although the nonamplified plasmid rescue procedure was site, rather than the Xho I site. Although little data will be effective, it was inefficient and necessitated the use of presented on the use of SVB, it can be transmitted and highly competent E. coli for transformation. A method was rescued as efficiently as the SVX vector. therefore devised to amplify, via replication, the covalently To generate highly transmissible virus carrying the SVX closed circles produced immediately following retrovirus genome, SVX DNA was transfected into $2 cells. The $2 infection. Plasmids that contain an SV40 origin replicate to cell line contains integrated copies of an M-MuLV provirus high copy number upon transfection of CCJSceils (Mellon genome that provides all the trans functions necessary for et al., 1981) a monkey cell line which expresses the SV40 the encapsidation of a recombinant genome, yet is defec- T antigen (Gluzman, 1981). Experiments were designed to tive in the ability to encapsidate its own RNA (Mann et al., determine if the SVX viral genome, which encodes an 1983). Eighteen hours after transfection, the #2 culture SV40 origin, could be introduced into cos cells and if the fluid was harvested and assayed for the presence of virions covalently closed circular products of reverse transcription capable of conferring G418 resistance (G418R) upon could be successfully amplified via SV40 T-antigen-medi- NIH3T3 cells. A titer of I-5 X lo3 CFU/ml was observed ated replication. For these experiments, a control plasmid, on 3T3 cells. To generate permanent cell lines that produce ZIPNeo, which lacked the SV40 origin but was identical to high titers of the SVX virus, transfected $2 cells were SVX in all other respects, was constructed and used to directly selected in G418 media. Several 42SVX lines generate a cell line (designated ZlPNeo 1A) via transient generated in this manner yielded viral titers ranging from rescue from transfected +2 cells. Both the SVX 2B and 1 O4 to over 1 O6 cfu/ml. ZlPNeo 1A cell lines were superinfected with a mixture of The structure of the SVX genome that is transferred to the amphotropic 1504A (Hartley and Rowe, recipient cells via virus infection was characterized by 1976) and the ecotropic virus, M-MuLV. The 1504A virus Southern blot analysis of DNA obtained from a random has been shown to infect a variety of mammalian cell lines, G418 resistant cell line, termed SVX 2B (Figure 3C). Diges- including mouse, monkey, rat, and human (Hartley and tion with Sac I (lane 2) as well as other restriction enzymes Rowe, 1976). The culture supernatant from SVX 28 was (Aha Ill, lane 7) confirmed the presence of a single proviral titered on both cos and CV-1 cells, and was found to have genome of the expected structure. Similar analyses of a titer of 2 x IO3 G418R cfu/ml and 3 X lo5 G418R cfu/ Murine Retrovirus Shuttle Vector 1055

Table 1. Summary of Rescue Efficiency and Activity No. of Clones

Rescue Helper Efficiency of Predicted Activity in Method Virus Vector Recoverf Structure Deletions Insertions Transient Rescueb

3T3 Infect. M-MuLV svx o-3 7 0 1 ~100% cm Fusron svx 2-5 x 103 14 2 2 ~100% El a(SVX) i&103 28 2 4 ND” SVB 5x IO3 14 2 2 ND cos Infect. 1504A svx Ice105 26 1 2 ~100% 1504A SVB IO4 17 0 0 ND 1504A + svx 6 35 0 ~100% M-MuLV aNumber of bacterial colonies (HBIOI LM1035) recovered from the Hirt supernatant prepared from 5 X IOs-IO7 mammalian cells, assuming that the competent have a transformation efficiency of 5 x IO’ colonies/pg plasmid (SVX) DNA. b Rescued plasmids and parental SVX (recircularized, single LTR plasmid) were transfected onto 42 and the transiently produced virus was harvested at 18 hr postglycerol shock. The supernatants were titered for G418 resistance on NIH 3T3 cells and the titer of the rescued plasmid was compared with the titer of the parental SVX. At least three individual rescued plasmids were tested in each case. ’ ND-Not determined.

ml, respectively. The culture supernatant from ZlPNeo IA 123456 7891011 was found to exhibit titers of 3 X 1 O4 G41 8R cfu/ml on cos cells and 9 X IO5 cfu/ml on CV-1 cells. Both viral genomes could therefore be successfully pseudotyped and intro- duced into cos and CV-1 cells, where they were also capable of neo gene expression. Examination of the inte- gration status of SVX in several independent cos lines indicated that SVX was not integrated and varied in copy number, from approximately 2 to 100 copies per cell (data not shown). The ZlPNeo virus was found to be integrated as a single copy provirus in several cos lines, as expected. The superinfected SVX 2B and ZlPNeo IA lines, pro- ducers of virus of both amphotropic and ecotropic host range, were then cocultivated with cos cells for up to 4 days. Hirt supernatants were prepared and examined for the level of SVX or ZlPNeo plasmid by Southern blot analysis and by transformation of E. coli HBI 01. A high level of SVX plasmid (5.0 ng/5 X IO6 cos cells) was Figure 2. Analysis of SVX Sequences Amplified during cos Cell-Mediated observed in the Hirt supernatant after 3 days of cocultiva- Rescue tion of SVX 2B and cos cells (data not shown). However, The DNAs indicated below were run on a 1 .O% agarose gel, transferred to the majority of the plasmids suffered deletions (see below). Zetabind paper (AMF/Cuno), and hybridized with a nick-translated neo Other cos cell infections were then set up, using as a probe according to standard procedures. A marker plasmid of SVX was prepared by cutting the original SVX vector with Sac I (which cuts once in source of virus clonal cell lines generated by infection of each LTR) and recircularizing the viral sequences by incubation with T4 SVX nonproducers with the amphotropic helper virus DNA ligase prior to transformation of E. coli HBI 01. The cloned plasmid, 1504A. When the Hirt supernatant was examined from one named SVX recirc, thus contained only one LTR and no proviral flanking of these infections, lo-fold more plasmid was obtained, cellular sequences or pBR322 sequences outside of those shown in Figure and very few molecules were seen to be deleted. (Figure I, (1) 50 pg of undigested SVX recirc; (2) 50 pg of SVX recirc digested wrth Bgl II; (3) 1 .O% of the Hirt supernatant from a IO cm dish of fused SVX 2, lanes 5-7). Preliminary experiments indicated that the 28 and cos cells, undigested; (4) Same as lane 3 but digested with Bgl II; level of SVX plasmid in cos cells reached a plateau after 3 (5) 0.1% of the Hirt supernatant from a IO cm dish of cos cells infected days of cocultivation. No ZlPNeo plasmid was observed in with SVX, undigested: (6) Same as lane 5 but digested with Bgl II; (7) Same the Hirt supernatant from cocultivation of cos and ZlPNeo as lane 5 but digested with Sac I; (8) 1 .O% of the Hirt supernatant from a IO cm dish of cos cells infected with ZlPNeo IA producers, undigested; IA (which produced virus that did not contain the SV40 (9) 1 .O% of the Hirt supernatant from a IO cm dish of cos cells fused with ) (Figure 2, lane 8). Bgl II digestion of ZlPNeo IA; (10) 1 .O% of the Hirt supernatant from a IO cm dish of unfused, the Hirt DNA obtained after cocultivation of the SVX pro- uninfected, cos cells; (11) 1.0% of the Hirt supernatant from a 10 cm dish ducer cells and cos cells revealed bands of 4.1 and 4.7 of unfused SVX 28 cells. kb (Figure 2, lane 6). The 4.1 kb band comigrated with the single LTR-SVX control plasmid, shown in lane 2, and the Hirt DNA with Sac I, which cuts once in each LTR, only higher MW band of 4.7 kb migrated at the position ex- one band of 4.1 kb was observed, suggesting that the pected for a plasmid with two LTRS. After digestion of the larger 4.7 kb DNA contained two LTRs (lane 7). Undigested Cell 1056

Hirt DNA electrophoresed as form I and form II molecules from cos-SV2B fusion yielded up to 5 x lo3 colonies from containing one or two LTRs, thus demonstrating that the 5 x IO6 cos cells. The structure of the majority of these amplified SVX plasmids did not exist as concatemers (lane plasmids was as predicted (single LTR-SVX molecules) 5). and the rescued plasmids were again active in a transient Transformation of E. coli HB101 with Hirt DNA obtained rescue assay (Table 1). Occasionally, plasmids that con- from cocultivation of 5 x 1 O6 cos cells with SVX producer tained insertions were isolated. Restriction mapping of cells (superinfected with 1504A alone) yielded up to IO5 several of these plasmids indicated that these molecules kanamycin sulfate-resistant colonies. The structure of plas- contained one LTR, were deleted at either the 5’ or 3’ end mids isolated from these colonies was analyzed by diges- of the SVX genome, and contained cellular sequences. tion with restriction enzymes, with the results summarized This structure is consistent with occasional recombination in Table 1. The majority of plasmids examined exhibited events that occurred between flanking cellular sequences the predicted SVX structure (see Table 1). However, if and SVX sequences. plasmids were isolated from SVX producers that were superinfected with both M-MuLV and 1504A, 85% of the Isolation of Cellular Sequences Flanking rescued plasmids contained deletions between the 3’ end Recombinant Proviruses of the LTR and the unique Barn HI site (data not shown). The studies of SV40 excision from rodent cells (Botchan Interestingly, this region was also a popular site for inser- et al., 1979, 1980) also indicated that in cell lines containing tions Restriction mapping of three randomly chosen plas- either a single SV40 genome or simply segments of the mids that contained insertions (including one plasmid SV40 genome containing the viral origin of DNA replication, which was rescued via NIH3T3 cell infection) revealed that fusion with CV-1 cells led to the excision and amplification insertion, as well as some deletion, occurred between the of a heterogenous population of molecules containing 3’ end of the LTR and the Barn HI site. The reason for this variable amounts of cellular DNA sequences adjacent to phenomenon is presently unclear. the site of integration of viral sequences. These observa- Amp/Bed Plasmid Rescue Following Fusion of SVX tions suggested that the site of recombinant retrovirus Nonproducer and Cos Cells integrations might be similarly obtained from the pool of Botchan et al. (1979, 1980) have shown that the fusion of free SVX molecules found in the Hirt supernatant following rodent cells carrying integrated copies of the SV40 genome PEG-mediated cos cell fusion. Southern analysis of the Hirt with monkey CV-1 cells leads to activation of the SV40 DNA from SVX 2B-cos cell fusions indicated that lO%- origin of replication in situ and the eventual excision and 15% of the excised plasmids contained putative inserts. amplification of closed circular SV40 DNA. In cells contain- To enrich for these molecules, Hirt DNA was fractionated ing multiple tandem copies of the SV40 genome, excision on a 1% agarose gel and the DNA isolated from the region appears to occur via homologous recombinational events, at the top of the gel was used to transform E. coli. since the majority of recovered viral genomes are unit Restriction analysis of plasmids rescued by this method length and infectious. Based on these studies, we deter- indicated that 60% of the DNAs contained inserts ranging mined whether the SVX proviral genome in SVX 2B cells from 5-8 kb and approximately half of the DNAs with could be similarly excised and amplified via fusion of the inserts contained two LTRs. Moreover, the site of insertions SVX 28 line with cos cells. Fusion between SVX 2B cells in the two LTR plasmids mapped between the LTRs, as and cos cells, or ZlPNeo 1A cells and cos cells, was expected for cellular sequences adjacent to the proviral accomplished by a brief exposure to polyethylene glycol site of integration (data not shown). 1000 (Davidson et al., 1976). After incubation of the fused Restriction mapping of five of these inserts indicated ceils for I, 2, or 3 days at 37°C Hirt DNA was prepared that the inserts were highly related, although not identical. and analyzed by Southern analysis, As shown in Figure 2, One clone, Fxl I, was further mapped with restriction the SVX 2B-cos cell combination resulted in 5 ng of enzymes and several fragments that contained exclusively predominately unit length closed circular SVX plasmid per cellular sequences were nick translated and used as 5 x IO6 cos cells. In contrast, the ZlPNeo 1A line, which probes for a Southern blot of uninfected NIH3T3 DNA. does not contain an SV40 origin, yielded no plasmid when Four of six fragments tested in this manner contained fused with cos cells (lane 9). After digestion of the SVX repetitive DNA. One Aha Ill fragment of 500 bp was Hirt DNA with Bgl II (which cuts once in the neo sequences identified as containing only single copy sequences, and of SVX), a band of 4.1 kb, which corresponds with the was therefore used as a diagnostic probe to analyze the control single LTR-SVX plasmid, was observed (lane 4). other two LTR-containing inserted plasmids (designated Thus the predominant recombination event that freed the Fxl plasmids) as well as DNA of NIH3T3 cells and the SVX plasmid from the flanking cellular DNA likely occurred SVX 28 cell line. As shown in Figure 3A, all four of the Fxl within the 600 bp direct repeat sequences of the LTR. plasmids not only contained sequences homologous to Alternatively, molecules that arose from recombination the putative integration site probe of clone Fxl I, but also events that occurred outside of the LTRs in the flanking retained the same Aha Ill fragment that was used as a cellular sequences (see below) efficiently recombined probe (lanes 2-5). As a control, SVX plasmid DNA was within the LTRs. also examined and found to be negative, as expected Transformation of E. coli HBI 01 with the Hirt supernatant (lane SVX). ~oyne Retrov~rus Shuttle Vector

I 2 3 4 5 6 7 8 9 10 I 2 3 4 5 6 7 8 9 10

- 1.0

- .5

Figure 3. Recovery of Cellular Sequences Flanking the Site of SVX integration via the cos Cell-Fusion Technique The cos fusion technique was used to generate plasmrds which were screened for the presence of insertions outside the LTRS (see Experimental Procedures). These plasmids, termed clones Fxl I-5, were analyzed by Southern blot analysis to determine if the inserted sequences were from the site of virus integration rn the lrne SVX 28. Plasmid Fxl 1 was digested with Aha Ill and a 500 bp fragment of entirely cellular sequence was nick translated and used to probe Southern blots of SVX recirc, Fxl 1-5, and cellular DNA from uninfected NIH3T3 and SVX 2B cells (A and B). (A) Analysis of Fxl plasmids cut with Aha Ill, (1) SVX DNA cut with Aha Ill. (2-5) Fxl plasmids cut with Aha Ill. (B) Analysis of uninfected NIH3T3 and SVX 28 cellular DNAs using the Aha Ill probe. (1) NIH3T3 DNA digested with Sac I; (2) SVX 28 DNA drgested with Sac I; (3) NIH3T3 DNA plus 10 pg of SVX recirc DNA digested with Sac I; (4) NIH3T3 DNA digested wrth Hind Ill; (5) SVX 28 DNA digested with Hind III; (6) NIH3T3 DNA drgested with Aha III; (7) SVX 28 DNA digested with Aha Ill; (6) NIH3T3 DNA plus 50 pg Fxl 1 digested with Aha Ill; (9) NIH3T3 DNA drgested with Pst I; (IO) SVX 2B DNA digested with Pst I. (C) Analysis of uninfected NIH3T3 and SVX 28 cell DNAs using Neo probe. The blot shown in (B) was washed with NaOH (according to manufacturer’s specifications) and reprobed with a virus-specific probe (the 1.4 kb neo fragment of SVX)

To further characterize the putative site of proviral inte- be seen that the recovered Aha Ill probe of Fxl 1 was in gration in SVX 2B cells, DNA from uninfected 3T3 cells fact identical in size to the homologous fragment in unin- and the SVX 2B cell line was cleaved with a variety of fected NIH 3T3 DNA. This confirms that the ligation of enzymes, transferred to Zetabind paper, and probed with flanking sequences did not occur within this fragment, the Fxl 1 Aha III fragment. If the probe was indeed from cellular DNA flanking the SVX provirus, a different Southern Generation of cDNA Copies of Genomic DNA by band pattern would be expected for line SVX 28 as Passage in the SVX Vector compared to NIH3T3 cells. As shown in Figure 38, lanes Passage of recombinant retrovirus genomes carrying DNA 1, 2, 4, 5, 9, and 10, digestion of SVX 28 DNA with Sac I, fragments which contained introns have been shown by Hind Ill, and Pst I yielded the expected heterozygous two other groups to result in the precise deletion of the pattern. (Heterozygous patterns were also obtained with intervening sequences, presumably via splicing of the Xba I and Bgl II, data not shown.) The blot shown in Figure retrovirus genome prior to encapsidatibn (Shimotohno and 38 was washed and reprobed with neo sequences to Temin, 1982; Sorge and Hughes, 1982). Since the SVX confirm that the “new” bands in the SVX 2B line, and vector affords a rapid means of recovering retrovirus ge- absent in NIH 3T3 DNA, contained the viral sequences. As nomes as bacterial plasmids, we explored the use of the shown in Figure 3B, the Hind Ill digest yielded positive vector to rapidly isolate cDNA copies of genomic DNA hybridization with neo where expected. As Sac I cleaved sequences. As a test case, the adenovirus 5 ElA region in the LTRs, the Sac I digest of SVX 28 simply liberated was chosen. This region is of interest in that it represents the intact SVX virus, which hybridized with neo, and co- a complex transcriptional unit which encodes several in- migrated with positive control plasmid added to NIH3T3 teresting functions implicated in viral and cellular transcrip- DNA (lanes 2 and 3). As shown in Figure 3B (lanes 6 and tional control (Berk et al., 1979; Jones and Shenk, 1980; 7) hybridization of Aha Ill digested NIH3T3 and SVX 2B Nevins, 1982; Schrier et al., 1983). In a typical lytic infection DNA with the cellular probe yielded the expected patterns of Hela cells, three distinct ElA mRNAs, designated 13S, of a single band of 500 bp, indicating that the probe was 12S, and 9S, are generated from a single transcript that is indeed entirely of cellular origin. If Fxl 1 plasmid DNA was processed by utilizing three different 5’ splice sites and added to NIH3T3 DNA and cut with Aha Ill (lane 8) it can one common 3’ splice site (Berk and Sharp, 1978; Chow Cell 1058

et al., 1979; Spector et al., 1978). Therefore, it was con- Barn HI ceivable that passage of the EIA sequences in SVX would Barn HI Xba I Xb+a I Sma I result in the generation of three distinct retrovirus genomes encoding the individual El A functions. The particular ade- novirus DNA fragment used for these experiments ex- tended from the Sac II site at nucleotide 356 to the Hpa I site at nucleotide 1574 (Tooze, 1981). The fragment there- fore contained the EIA promoter (but not enhancer) and the entire coding region for the 9, 12, and 13s RNA encoded proteins, but lacked the ElA polyadenylation site. After introduction of the ElA containing fragment into the Barn HI site of SVX, the recombinant DNA was transfected into the packaging line, #2; a transient harvest was taken 18 hr later, and the supernatant used to infect NIH3T3 cells. G418R colonies were selected, isolated, and ex- panded in order to analyze and eventually recover the El A-retrovirus genome. Southern blot analysis of Barn HI and Xba I digests of DNA from several independent colo- nies indicated that the EIA insert in each case had under- gone a specific deletion that corresponded in size to that expected for the 13s EIA mRNA (data not shown). No EIA inserts corresponding to unspliced EIA or the two other known EIA splices were found. In order to confirm Figure 4. Restriction Enzyme Analysis of El A-SVX Proviral DNAs Re- covered from Fusion of El A-SVX-Infected NIH3T3 Clonal Lines and cos the results of the Southern blot analysis, several independ- Cells ent colonies were fused with cos cells as described above Lane 1 contains DNA from the starting plasmid; lanes 2 and 3 contain DNA and Hirt DNA was isolated and used to transform compe- from two representative rescued plasmids isolated from fusion of two tent E. coli HBI 01. Digestion of several rescued plasmid independent NIH3T3 colonies. genomes with the diagnostic enzymes Sma I, Barn HI, Xba I, and Barn HI plus Xba I confirmed the results seen on the Xba I). The .98 kb fragment encodes the splice donor Southern blot (Figure 4). sequences, so a 13S, 12S, or 9s splicing event should As Barn HI was the unique site into which EIA was reduce the size of this fragment to .87 kb, .73 kb, or .47 inserted, digestion of the original El A-SVX plasmid with kb, respectively. As shown in Figure 4, lanes 2 and 3, the Barn HI resulted in a 1.2 kb El A fragment and a 10 kb expected band of .87 kb was observed. One further digest, vector backbone fragment (Figure 4, lane 1, Barn HI Sma I, confirmed the 13s structure of the rescued plas- digest). The splice donor for a 13s splice is located at mids. Sma I cuts once in the LTR, once at the 5’ end of adenovirus nucleotide number 1111, while the 12s and 9s the inserted ElA fragment (an artifact of the construction donors are located at nucleotides 973 and 636, respec- method), once within EIA at adenovirus nucleotide number tively (Svensson et al., 1983). The common 3’ acceptor is 1010, once near the 3’ end of the Barn-Xho fragment of at nucleotide number 1226. Therefore digestion of rescued SVX which encodes the 3’ splice site of M-MuLV, and plasmids with Barn HI should yield a 1 .l kb, .96 kb, or .71 once near the 3’ end of the neo gene. Digestion of the kb El A-containing fragment if a 13S, 12S, or 9s splicing original El A-SVX with Sma I resulted in fragments of 6 kb, event has occurred. As shown in lanes 2 and 3 (Barn HI 0.5 kb, 0.15 kb, 0.92 kb, 1.3 kb, and 1.9 kb. As the 0.92 digest), the expected 1.1 kb EIA fragment diagnostic of kb fragment contains the 5’ donor sequence for a 13s the 13s cDNA was observed. The fragment of 4.1 kb was splice, this fragment should be reduced to .80 kb after derived from the vector backbone. Xba I cuts twice in the Sma I digestion (Figure 4, lanes 2 and 3, Sma I digest). A El A-SVX proviral DNA, once in the middle of the LTR and 12s or 9s splice would eliminate the Sma I site at 1010 once in EIA sequences at adenovirus nucleotide number nucleotides, and thus eliminate both the .92 kb and .65 kb 1340. The original El A-SVX construct contained two LTRs fragment. and therefore yielded three fragments of 6.0 kb, 1.7 kb, Several rescued plasmids were subsequently se- and 3.5 kb upon Xba I digestion (Figure 4, lane 1, Xba I). quenced and found to contain precise DNA copies of the The rescued plasmids, which contained a single LTR and 13s ElA mRNA. Retrovirus genomes containing DNA a 13s cDNA, yielded fragments of 3.5 kb (containing copies of the 12s and 9s mRNAs have been similarly primarily vector sequences) and 1.6 kb. Since the 1.7 kb obtained by passage of the unspliced ElA virus through Xba I fragment of the original El A-SVX contained the 13s Hela cells (B. E. Roberts et al., unpublished data). splice donor, the reduction in size to 1.6 kb was as expected for a 13s cDNA. Digestion of the original El A- Discussion SVX plasmid with Barn HI plus Xba I yielded fragments of The SVX shuttle vector described here provides a powerful 6 kb, .98 kb, and .23 kb (Figure 4, lane 1, Barn HI and system for both the introduction of genes into mammalian l$y$e Retrovirus Shuttle Vector

cells and the whole animal and eventual recovery of the However, one important difference between our protocol transferred sequences as molecular clones. In construction for generating virus from vector DNA and previously re- of the vector, all of the &-acting sequences necessary ported strategies is that we have exploited the high tran- for the efficient transmission of the viral genome have been sient expression of the vector shortly after transfection to retained. Generation of highly transmissible virus carrying increase the representation of cloned DNAs as infectious the appropriate recombinant genome is made possible by virus. Although the SVX vector per se yields only approxi- transfection of the SVX DNA into cells that provide essential mately lo3 G418’ Cfu/ml, we have previously shown that virus gene products (Mann et al., 1983). Expression of the incorporation of a plasmid backbone containing the sequences inserted into SVX is achieved through the use polyoma virus early region permits the rescue of over IO5 of the normal retrovirus transcriptional signals, which give cfu/ml (Mulligan, 1983). This efficiency of rescue will be rise to both genomic length and spliced viral transcripts. crucial for the eventual use of the system for gene isolation PO date, we have introduced over a dozen sequences studies. (both cDNA and genomic) into the vector and have dem- All of the rescue protocols described here result in the onstrated both transcriptional and translational expression rescue of proviral genomes exhibiting both the proper in a majority of cases. Although the size constraints upon structure and high biological activity upon retransfection. the inserted sequences have not yet been rigorously de- The most facile means of rescuing a recombinant genome fined, we have introduced sequences as large at 4.4 kb is probably the cos fusion method. This method does not into SVX without perturbation of transmission or splicing. rely on the superinfection of the transformant of interest Based on the occurrence of variant M-MuLV genomes as with virus, and results in thousands of bacterial transform- large as 11 kb (S. P. Goff, personal communication), we ants harboring the desired proviral DNA. Nevertheless, the suspect that 6.0 kb will represent the upper limit of insert procedure does require the selection and expansion of a sizes. However, since the size limitation applies only to the transformant prior to fusion. For certain applications, par- encapsidated genome, it is likely that large genomic inserts ticularly in cases where creation of cDNAs from genomic (in the 20 kb size range) could be introduced into the DNA sequences is desired, it may be possible to shorten vector for the purpose of intron deletions as long as the the process significantly. For example, we are currently truncated insert is less than 6 kb in size. Experiments to testing whether it is possible to rescue proviral genomes test this prediction are in progress. by simply infecting cos cells with the small amount of virus In addition to size, there do appear to be several other harvested from a transient-rescue assay. factors that can affect transmission of a recombinant The ability to recover recombinant retroviral genomes genome. Shimotohono and Temin (1981) reported that the and adjacent chromosomal sequences rapidly has impli- presence of polyadenylation sites within an insert can cations beyond application to gene isolation schemes. We drastically reduce transmissibility, presumably because of are particularly interested in applying the system towards the generation of prematurely polyadenylated transcripts studies of the effects of chromosomal position upon the that lack the capacity to be properly replicated. In the one expression and regulation of genes transferred into cells instance in which we have inserted a sequence with a in vitro and in vivo via retrovirus-mediated transduction. poly(A) site (the polyoma large T cDNA sequence), no The ability to rapidly isolate sequences flanking proviral reduction in G418 titer from that of SVX itself was detected integration sites will provide a source of probes for exam- (C. Cepko, P. Jat, P. Sharp, and R. Mulligan, unpublished ining adjacent chromosomal sequences in a number of observations). Since the rules that govern the preferential ways (e.g., transcriptional activity, DNA methylation, utilization of a particular poly(A) site when situated adjacent DNAase hypersensitivity, etc.). In addition, as to several additional poly(A) sites have not yet been de- have been observed to behave as insertional mutagens fined (Horowitz et al., 1983) the effects of various poly(A) (Varmus et al., 1981; Jenkins et al., 1981; Jaenisch et al., sites on the generation of full-length genomic RNA will 1983) as well as activators of gene expression (Hayward likely be variable. et al., 1981; Payne et al., 1982), SVX integrations that We have also found that the insertion of a few se- create a selectable alteration in the expression of nearby quences into SVX appears to affect drastically the gener- sequences can provide flanking sequence probes for the ation of the subgenomic viral RNA that encodes G418 gene under study. Another interest is to use the shuttle resistance. In such cases, the few G418R colonies that features of the vector to characterize the spectrum of can be isolated contain rearranged proviral genomes (A. mutations induced as a consequence of retrovirus repli- Korman and R. C. Mulligan, unpublished data). To alleviate cation this problem, we have constructed vectors that lack the The most useful practical application of the system at retroviral 5’ and 3’ splice sites and contain independent present, however, is probably the rapid isolation of cDNA transcriptional signals for promotion of selectable marker sequences from genomic DNAs containing intervening expression. sequences. In particular, in cases where the site of synthe- Similar strategies for exploiting retroviruses as vectors sis of an mRNA is not known, a corresponding genomic have been reported by a number of laboratories (Shimo- DNA sequence can be isolated, or in the case of genes tohno and Temin, 1981; Wei et al., 1981; Tabin et al., encoding large mRNAs, the shuttle rescue procedure may 1982; Miller et al., 1983; Joyner and Bernstein, 1983). offer the most rapid means of generating full-length cDNA Cell 1060

sequences. We have already utilized the vector to isolate Protocols for Rescue of Proviral DNA cDNA copies of a variety of genomic inserts, including the For rescue of nonamplified SVX, producer cell lines were cocultivated with uninfected NIH3T3 cells at a ratio of I:4 (approximately I-5 X IO6 cells/ adenovirus ElA, adenovirus EIB, and SV40 T-antigen- 100 cm2 dish) in the presence of 2 pg/ml polybrene for 20 hr. A Hirt coding regions (in collaboration with P. Jat and P. A. Sharp, supernatant (Hirt, 1967) was then prepared, extracted once with M.I.T.) and human DR sequences (A. Korman et al., un- phenol:chloroform (1 :I ratio) and once with chloroform. The aqueous phase published data). Similar success in isolating cDNA se- was precipitated with isopropanol at -20°C at least twice. The final pellet was resuspended in a minimum volume of 10 mM Tris (pH 7.4) and 1 mM quences has been obtained in M. Botchan’s laboratory EDTA. E. coli HBIOI (strain LM 1035, a gift from Y. Nagamine, Rockefeller (University of California at Berkeley). Our recent studies of University) were then transformed directly with this preparation (Lederberg adenovirus EIA function which have utilizted the SVX and Cohen, 1974) or the preparation was digested for analysis by the vector for the isolation of individual EIA cDNAs suggest Southern blotting procedure. In preparation of competent E. coli HBIOI for that the system may also be generally useful for dissecting transformation, the bacteria were harvested at 00, = 0.2 and the heat shock step was carried out at 37°C per Y. Nagamine (personal communi- complex Jtranscriptional / units /encoding Jdifferentially cation). Previously frozen (Morrison, 1979) competent LM 1035 cells yielded spliced, overlapping transcripts (B. E. Roberts et al., un- 5 x 107-10’ transformants per pg of pBR322. published data). For rescue via cos cell infection, a line of SVX-infected cells was The generality of the method for isolating cDNA se- superinfected with 1504A virus. After allowing 1-2 weeks for spread of 1504A through the culture, the SVX producers were split 1:iO onto cos quences from genomic inserts, however, needs to be cells, split from a confluent plate the day before and the two cell types further established by examining the fate of additional were cocultivated for 3 days in DMEM plus 10% calf serum, 10% fetal calf inserts. In particular, the finding that only 13s cDNA-SVX serum, and 2 *g/ml polybrene. Media was changed after 1 and 2 days. A genomes are generated after transfection of #2 cells with Hid extract was then prepared as described above. the El A-SVX recombinant DNA, yet all three cDNA species For fusion rescue, SVX 28 and cos cells were plated at a 1 :I ratio by splitting both cell types from newly confluent dishes at a I:2 ratio into are generated after transfection of Hela cells, suggests Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal calf that an added complexity exists in the system. Although serum and 10% calf serum. When the cells were very confluent (12-48 hr the results may reflect a species or cell type difference in after plating), they were washed twice with DMEM and exposed to a 50% the efficiency of the various splicing reactions, we have polyethylene glycol 1000 (Baker) solution in DMEM (50% v/v), for 60 set recently shown that the infection of 3T3 cells with Ad5 at room temperature (Davidson et al., 1976). The cells were then quickly washed twice with DMEM and twice with DMEM plus 10% calf serum. The virus results in the production of both 135 and 12s mRNAs. fused cells were incubated in DMEM containing 10% calf serum and 10% It is therefore likely that the differences observed using the fetal calf serum for 3 days, with media changes every day. Hirt DNA was retrovirus constructs in part reflect inherent differences in then prepared and used to transform HBIOI as described above. either the structure of chimeric retrovirus-El A primary tran- For recovery of flanking sequences at the site of virus integration, cos cells were fused with line SVX 28 as described above. The resulting Hirt scripts or the kinetics of transport of unspliced recombinant supernatant from a 100 cm2 dish of fused cells was fractionated by retrovirus RNA versus normally spliced Ad5 RNA. Experi- electrophoresis on a 14 cm 1 .O% agarose gel. After running the bromphenol ments to test a variety of additional genomic inserts, as blue dye to the bottom of the gel, a 1-2 cm wide slice of the gel just below well as to resolve the different patterns of cDNA recovery the sample well was removed (high molecular weight DNA of the isolated observed in mouse and human cells, are underway. slice ran just ahead of the selected slice). The DNA in the slice was isolated by the glass bead procedure (Vogelstein and Gillespie, 1979) and used to transform E. coli. Experimental Procedures Acknowledgments Cell Culture, DNA Transfections, and Rescue of Recombinant Virus We thank the other members of our laboratory for continual helpful discus- NIH3T3 cells, obtained from G. Cooper (Dana-Farber Cancer Institute), CV- sions. We are grateful to Y. Nagamine for his gift of HBIOI LM1035 and 1 cells, and $2 cells (Mann et al., 1983) were maintained in Dulbecco’s helpful tips on high efficiency bacterial transformation. This work was Modified Eagle’s Medium (DMEM) supplemented with 10% calf serum. M- supported by grants from the National Cancer Institute and the MacArthur MuLV virus was obtained from the clone 1 cell line (Fan and Paskind, 1974), Foundation. C. L. C. is a Jane Coffin Childs Postdoctoral Fellow. and 1504A virus (Hartley and Rowe, 1976) was a giff from Nancy Hopkins The costs of publication of this article were defrayed in part by the (M.I.T.). A subclone of cos cells, cos M6, isolated by M. Horowitz (Horowitz payment of page charges. This article must therefore be hereby marked et al., 1983) from cos-I cells of Y. Gluzman (1981) (Cold Spring Harbor) “advertisement” in accordance with 18 U.S.C. Section 1734 solely to were matntained in DMEM and 10% fetal calf serum. Transfections were indicate this fact. via the CaP04 technique of Graham and van der Eb, as modified by Parker and Stark (1979). To rescue recombinant SVX virus, approximately 5 x 1 O5 $2 cells (seeded 24 hr earlier) were transfected with IO pg SVX DNA. Received March 8, 1984; revised May 2, 1984 Eighteen hours after the glycerol shock, culture media was collected and filtered through a .45p Nalgene filter. To infect cells, 1 ml of the supernatant References was added to 5 x IO5 NIH3T3 cells in a 100 cm* dish and incubated for 2 hr at 37°C in the presence of 8 Mg/ml polybrene (Aldrich). Complete media Berk, A. J., and Sharp, P. A. (1978). Structure of adenovirus 2 early mRNAs. was added, the cultures were incubated for 2-3 days, and were then split Cell 14, 695-711. into complete media containing 1 mg/ml G418 (Gibco) (Davies and Jime- Berk, A. J., Lee, F., Harrison, T., Williams, J., and Sharp, P. A. (1979). Pre- nez, 1980). early adenovirus 2 gene product regulates synthesis of early viral messenger RNAs. Cell 7 7, 935-944. 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