General strategy for constructing large HSV-1 plasmid vectors that co-express multiple genes

Abstract

Herpes simplex virus type 1 (HSV-1) plasmid vectors have a number of attractive features for gene transfer into neurons. In particular, the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to co-express multiple genes. Here, we report a general strategy for constructing large HSV-1 plasmid vectors that co-express multiple genes. Each transcription unit is linked to an antibiotic resistance gene, and genetic selections are used to assemble large vectors. Using this strategy, we constructed large (26 or 31 kb) HSV-1 vectors that contain two transcription units and two or three genes. These vectors were efficiently packaged into HSV-1 particles using a helper virus-free packaging system. The resulting vector stocks supported the expression of two or three genes in both cultured cells and the rat brain. Potential applications of HSV-1 vectors that co-express multiple genes are discussed.

Figure 1. Diagram of the strategy for constructing HSV-1 vectors that contain multiple genes

An individual gene plasmid contains an EcoRV site for inserting an antibiotic resistance gene, a polylinker (XhoI, PvuII, HindIII, SphI, PstI, AccI, SalI, XbaI, BamHI, SmaI, KpnI, SacI, and EcoRI) for inserting a promoter and gene, an intron and poly A site, a HpaI site for inserting boundary elements such as a MAR, and the entire array is flanked on each end by an 8-bp site for an endonuclease (FseI in the example shown). An 8-cutter plasmid contains four sites (FseI, PmeI, SwaI, and SgrAI) to receive individual transcription units (inserted using a selection for the specific antibiotic resistance gene), a polylinker (XhoI, PvuII, HindIII, SphI, PstI, AccI, SalI, XbaI, BamHI, HindIII, and BglII) for the direct insertion of a transcription unit, and this array is flanked on each end by PacI, AscI, and NotI sites to excise the array of transcription units. An HSV-1 vector backbone plasmid contains an HSV-1 a sequence (packaging site), PacI, AscI, and NotI sites to insert an array of transcription units using a selection for a specific antibiotic resistance gene, and an HSV-1 origin of DNA replication HSV-1 ori_s). The HSV-1 ori_s fragment contains an HSV-1 IE 4/5 promoter that is separated from the PacI, AscI, and NotI sites by three poly A sites (TriA) (12). The pBR sequences (horizontal lines) in each plasmid contain the amp^r gene and the ColE1 origin of DNA replication. F, FseI; R, EcoRV; H, HpaI; P, PacI; A, AscI; N, NotI; Pm, PmeI: Sw, Swa1: and Sg, SgrAI.

Figure 2. TH-IR, AADC-IR, and X-gal-positive striatal cells in a rat sacrificed four days after microinjection of pTH-NFHth/ires/aadc\TH-NFHlac(D;l-)

This TH contains the HA tag (13) and was detected using an anti-HA antibody. AADC was detected using an anti-human AADC antibody, β-gal was detected with X-gal (16). (A and B) Low- and high-power views of TH-IR. Numerous positive cells are visible, and many display neuronal morphology. (C and D) Low- and high-power views of AADC-IR. (E and F) Low- and high-power views of X-gal-positive cells. Scale bars: A and E, 50 μm; B and F, 25 μm; C, 25 μm; and D, 25 μm.

Figure 3. Flag-IR and β-gal-IR are present in the same postrhinal cortex cells in a rat sacrificed four days after microinjection of pTH-NFHpkΔ\TH-NFHlac (D;l-)

PkcΔ contains the flag tag (16) and was detected using an anti-flag antibody that was visualized using a rhodamine-conjugated secondary antibody. β-gal was detected using an anti-β-gal antibody that was visualized using a fluorescein-conjugated secondary antibody. (A–C) Low-power views of the same field showing a number of cells that contain (A) β-gal-IR and (B) flag-IR. (C) A double exposure shows co-stained cells. (D–F) High-power views of the same field showing one large cell with a pyramidal-shaped cell body and a process that resembles an apical dendrite. This cell contains (D) β-gal-IR and (E) flag-IR. (F) A double exposure shows that this cell is co-stained. Scale bars: A–C, 25 μm and D–F, 25 μm.