Construction and characterization of a replication-competent retroviral shuttle vector plasmid

Abstract

We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.

FIG. 1.

Schematic drawing showing the structure of the RSVP vector. (A) RSVP. The viral genes gag, pol, and env are shown (not to scale). The positions of the splice donor (SD) and SA are also shown. (B) Schematic representation of the cassette. EM-Bsd, EM-7 promoter-blasticidin resistance gene; EM-Zeo, EM-7 promoter-zeocin resistance gene; lacO, lac operator.

FIG. 2.

Rescue of unintegrated retroviral DNA. Low-molecular-weight DNA was isolated by HIRT extraction and the extracted DNA was used to transform E. coli DH5α. The selection was for either blasticidin- or zeocin-resistant colonies. (A) Diagram showing the difference between two-LTR and one-LTR DNA segments. (B and C) Plasmids were digested with ClaI and SacI. The positions of DNAs containing two LTRs and one LTR are indicated. C, parental RSVP vector; M, size marker (λ DNA digested with HindIII).

FIG. 3.

Rescue of integrated retroviral DNA. (A) Schematic diagram of lac repressor-mediated recovery (see Materials and Methods for details). (B) Plasmids were digested with ClaI and MluI. The position of the cassette insert is indicated. C, parental RSVP vector; M, size marker (λ DNA digested with HindIII).

FIG. 4.

Sequences of integrated retroviral DNA. The inverted triangle indicates the boundary between the two LTRs. Sequences flanking the LTR are shown in bold.

FIG. 5.

Major by-product obtained in the recovery of integrated viral DNA. (A) Schematic drawing of the generation of the plasmid. M, MluI; B, BamHI; C, ClaI; C*, ClaI site within gag gene subjected to dam methylation in E. coli but not in avian cells. (B) Restriction enzyme analyses of the plasmid. Digestion with ClaI did not cut the plasmid due to dam methylation. Digestion with MluI cut the plasmid once and digestion with BamHI generated two fragments, of 1.54 and 1.39 kb, as expected. kb, DNA ladder in kilobases.

FIG. 6.

Southern transfer analyses of the stability of the RSVP vector. (A) The probe was prepared from RCASBP(A) as a 1.2-kb EcoRI fragment to provide equal opportunity to hybridize to EcoRI fragments that contained (or had lost) the cassette. EcoRI recognition sites are indicated (E) (not to scale). (B) Detection of the cassette insert in the genomic DNA derived from cells infected with the RSVP(A)B and RSVP(A)Z vectors. Genomic DNA was digested with EcoRI, resolved in an agarose gel, transferred onto a nitrocellulose membrane, and hybridized with ³²P-labeled DNA prepared from the 1.2-kb EcoRI fragment of RCASBP(A). The larger band represents a 2.4-kb EcoRI fragment containing the insert, whereas the smaller band represent a 1.2-kb EcoRI fragment lacking the insert.