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. 2008 Nov;237(11):3260-9.
doi: 10.1002/dvdy.21766.

Murine retroviruses re-engineered for lineage tracing and expression of toxic genes in the developing chick embryo

Sara J Venters  1 , Magnus R Dias da Silva, Jeanette Hyer
Affiliations

Murine retroviruses re-engineered for lineage tracing and expression of toxic genes in the developing chick embryo

Sara J Venters et al. Dev Dyn. 2008 Nov.

Abstract

We describe two replication incompetent retroviral vectors that co-express green fluorescent protein (GFP) and beta-galactosidase. These vectors incorporate either the avian reticuloendotheliosis (spleen necrosis virus; SNV) promoter or the chick beta-actin promoter, into the backbone of the murine leukemia (MLV) viral vector. The additional promoters drive transgene expression in avian tissue. The remainder of the vector is MLV-like, allowing high titer viral particle production by means of transient transfection. The SNV promoter produces high and early expression of introduced genes, enabling detection of the single copy integrated GFP gene in infected cells and their progeny in vivo. Substitution of the LacZ coding DNA with a relevant gene of interest will enable its co-expression with GFP, thus allowing visualization of the effect of specific and stable changes in gene expression throughout development. As the VSV-G pseudotyped viral vector is replication incompetent, changes in gene expression can be controlled temporally, by altering the timing of introduction.

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Figures

Figure 1
Figure 1. pACID and pSNID vectors
Graphic representation of relevant features of the proviral vector cloning and resulting virion. The proviral genome is derived from the Murine Leukemia Virus (MLV; light blue), with a second promoter (orange) added downstream of the 5’ LTR. The use of two shuttle vectors significantly aids in cloning and virus design and allows for facile swapping of genes of interest. pSNID and pACID contain two reporter genes (GFP and LacZ). To produce virus, the proviral plasmid and a plasmid containing the VSV-g envelope protein are transfected into the Phoenix packaging cell line, a 293 cell line that has stably incorporated the genes for MLV gag and pol (nuclear light blue). The provirus is packaged into a gag-derived protein coat along with polderived enzymes (blue triangles) and is released into the culture supernatant. The VSV-G protein is inserted into the membrane coat of the budded virus, and acts as a receptor for lipid moieties in target cells, which confers a broad range of infectivity.
Figure 2
Figure 2. pACID and pSNID are expressed in all targeted embryonic tissues
A–D: LacZ expression in embryos infected with 1–5nl of concentrated pACID (3.1×105) or E–H: pSNID (1.4×106) viral supernatant. Embryos were infected at between HH stage 9 and 12 (8 to 18 somite pairs) by targeted retroviral microinjection and re-incubated between 2 to 5 days. LacZ expression is evident in the eye (A and E, arrows), extra-ocular mesenchyme and head mesoderm (E, arrowheads), telencephalon (B and F, arrows) following targeted injection into the optic vesicle, head mesoderm and forebrain respectively. Infections targeted to the posterior neural plate gave rise to columnar clones in the resulting neural tube (C, arrows). Injection in the lateral plate mesoderm and heart field resulted in infected cells in the forelimb and heart (G). Expression is seen in somite compartments following presomitic mesoderm infection (D and H). LacZ expressing cells are in the sclerotome (D) and in the dermomyotome (H, arrow) and myotome (H, arrowheads). pACID LacZ expression is shown in cleared embryos (A, C, and D). I–L: LacZ expressing cells following targeted infection with 100nl of concentrated pSNID virus supernatant (7.2×107) and 4 days reincubation. I: Infected cells in the first branchial arch (arrow) and periocular mesenchyme (arrowhead). Stained cells are also evident in the eye. J: Sagittal section through the embryo shown in I. Multiple LacZ expressing cells are evident in the nasal portion of the branchial arch. K: Heavily infected mesoderm following infection at the epithelial somite stage. L: Transverse section through the embryo shown in K. Infected cells are seen throughout somitic derivatives on the infected side. t; telencephalon, FL; forelimb, h; heart, NT; neural tube, BA; branchial arch, nc; notochord.
Figure 3
Figure 3. GFP and LacZ are robustly co-expressed at 48 hours post-infection from pSNID
A–F: Marker expression following targeted injection of 5nl pSNID concentrated supernatant (1.2×107). Direct GFP epifluorescence (A, C, and E) and corresponding LacZ expression (B, D, and F) in the same embryos 48 hours after infection. Expression of both markers is evident in eye, brain, and head mesoderm (A and B) after targeting the head mesoderm, optic vesicle, and forebrain in a stage 11 (14 somite) embryo. C and D: GFP and LacZ expression in the myotome (arrows) and dermomyotome of an embryo where the presomitic mesoderm was infected at stage 10 (10 somites). D and E: Expression of GFP and LacZ in the heart of an embryo infected at stage 12 (16 somites). G and H: Sections of embryos similarly injected with concentrated pSNID supernatant. G: LacZ expressing neural crest cells are evident around the eye. H: A small clone of LacZ expressing cells in the myocardium of the heart. FL; forelimb, h; heart, OT; optic tectum.
Figure 4
Figure 4. pSNID-driven markers are detectable less than 24 hours after infection but pACID-driven markers require amplification
A and B: Infection resulting from injection of pSNID viral supernatant into epithelial somites with direct visualization of GFP (A), followed by staining for LacZ expression (B). Both markers expressed in the same somite cells 18 hours after infection (arrows). C: GFP expression in somites (arrows) and lateral plate mesoderm (arrowheads) 12 hours after infection with pSNID. D: pSNID driven marker expression in the dermomyotome at 8 hours post infection of the somite. E: GFP expression in the optic cup of an embryo 24 hours after electroporation with the pACID proviral vector. F: GFP expression in the neural tube of an embryo electroporated with the pSNID vector 24 hours previously. G–I: Immunohistochemical localization of beta-galactosidase (H) and GFP (I) and the merged images (J) in the telencephalon of a pACID infected embryo 3 days after infection. OC; optic cup, s; somite, t; telencephalon, NT; neural tube.
Figure 5
Figure 5. Marker expression is persistent through several days of embryonic development
A–C: LacZ expression in embryos infected with 5nl of pACID (2.2×106) between HH stage 10–13 and re-incubated to E7. A: LacZ expressing cells in the forelimb after targeting infection to forelimb lateral plate mesoderm. B: LacZ expressing cells in the pigmented epithelium domain of the eye 6 days after infection at stage 12. C: Clusters of LacZ expressing cells in the heart and forelimb (arrows) 5 days after infection. D–E: LacZ expression resulting from infection with 5nl of pSNID (2.9×106). D: LacZ expressing cells in the ectoderm 6 days after pSNID infection. E: Infected cells in the pigmented epithelium of the eye (arrow) 5 days after targeted infection. F: GFP expression in neural crest cells (arrows) extending over the eye 6 days after infection at the 13 somite stage. FL; forelimb, h; heart, L; lens.
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