Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 - new vectors for in vitro and in vivo delivery

Abstract

Background: Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells.

Results: We describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks.

Conclusion: The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.

Figure 1

Maps of the retrofitting vectors, BACs and the retrofitted BACs a) pRetro vectors; pRetroNeo, pRetroNeoLuc, pRetroNeoLucOE and pRetroNeoOE. Selected restriction sites are indicated. b) Retrofitting of standard BACs; pBeloBAC11, pBeloBAC11 retrofitted with pRetroNeo (pBeloBACRetroNeo), pBACe3.6, and pBACe3.6 retrofitted with pRetroNeoLucOE (pBACe3.6RetroNeoLucOE). NotI sites are indicated. c) Retrofitting of specific BACs; BACLucA, BACLucARetroNeoOE, BACGFPNeoOE, and BACGFPNeoOERetroNeoLuc. NotI sites are indicated. The following elements are labelled; spectinomycin resistance gene (SpR), gamma origin from the R6K plasmid (R6K ori), loxP site (LoxP), G418 resistance cassette (NeoR), luciferase expression cassette (Luc), oriP from Epstein Barr virus (OriP), EBNA-1 gene from Epstein Barr virus (EBNA-1), chloramphenicol resistance gene (CmR), F1 origin from the F factor (F1 ori), SacBII gene (SacBII), lacZ gene (LacZ) and EGFP expression cassette (eGFP).

Figure 2

In vivo retrofitting of BACs a) The pRetro plasmids are cotransfected with a Cre-expression plasmid, pJM2545, into the BAC-containing E. coli. PJM2545 carries a temperature sensitive origin of replication (rep^tc) kanamycin resistance (Km^R) and an inducible cre gene (Cre). Recombination between the loxP sites (LoxP) results in the retrofitted BACs which carry both chloramphenicol and spectinomycin resistance (Cm^R and Sp^R). b) Pulsed-field gel showing retrofitted BACs. BAC DNA was digested with NotI and separated on a pulsed-field gel. Lane 1) BACLucA, 2) BACLucARetroNeoOE, 3) BACGFPNeoOE and 4) BACGFPNeoOERetroNeoLuc. M indicates the Low Range PFG Marker from Biolabs. Sizes of the markers are indicated on the right. c) Pulsed-field gel showing BAC 4B19 retrofitted with pNELγ. DNA from BAC 4B19 (BAC) and the BAC retrofitted with pNELγ (BACNEL) was digested with NotI and separated on a pulsed-field gel. L indicates the 1 Kb DNA Ladder from GibcoBRL and M indicates the Low Range PFG Marker from Biolabs. The sizes of the markers are indicated on the right.

Figure 3

Transient transfection showing the luciferase expression measured at 24 hours Plasmid (1 μg) and BAC (3 μg) DNA as indicated below the bars was transfected into the cells with PEI22. Two separate experiments were carried out. Each experiment was done in triplicate and the error bars indicate the standard deviation.

Figure 4

Analysis of stable cell lines 0.5 μg of BACGFPNeoOE DNA was transfected into B16F10 cells with Lipofectamine 2000 and 6 clones were expanded in G418 selection. a) The percentage of cells expressing GFP was determined by FACs analysis at 30 days. B16F10 indicates untransfected cells. b) Analysis of DNA rescued from the 6 stable cell lines. DNA was extracted from the 6 cell lines and re-transfected into E. coli. DNA from 3 E. coli clones are shown for each B16F10 cell line as indicated above the lanes. Cell line number 2 gave rearranged BAC DNA in two E. coli clones, while the third is unrearranged. DNA was cut with SalI and resolved on a 0.3% SeaKem Gold agarose gel. Size marker M1 is the 1 kb DNA Ladder from Promega and M2 is the Lambda DNA-Mono Cut Mix from Biolabs. In between the two markers is the input BAC DNA cut with SalI. The sizes of the markers and BAC fragments are shown on the left and right of the gel respectively. c) Pulsed-field gel showing that the BAC DNA rescued from the cell lines is the same as the input DNA. The lanes carry; Low Range PFG marker (Biolabs)(M), input BACGFPNeoOE DNA cut with SalI (input), and DNA from three E. coli clones from cell line number 4 (4a, 4b and 4c) cut with SalI. The sizes of the markers and BAC fragments are indicated on the left and right of the gel respectively.

Figure 5

In vivo delivery and expression of luciferase Twenty μg each of plasmid pCMV-LUC and BACs BACLucA, BACLucARetroOE, and BACGFPNeoOERetroNeoLuc DNA was injected and electroporated into mouse muscle. Luciferase expression was assayed 24 hours after gene delivery. Background levels in un-treated mice is about 50 RLU.