Retroviral vectors are useful tools for gene transfer into cells either in vitro or in vivo (Figure 1) . They enable the stable integration of a transduced gene into the host chromosome via the integration mechanisms that they normally employ during their normal life cycle. We have been designing and testing several types of retroviral vectors for use in mammals and chicks.

MURINE RETROVIRUS VECTORS. The murine vectors have been useful for lineage tracing in rats and mice. We have designed several vectors that encode a histochemical marker gene and a library of oligonucleotide tags (Golden et al. 1995) . Our most recent such vector is the BOLAP library LINK. All of our libraries are freely available.

We have made several vectors which are useful for the expression of a gene of interest and a histochemical reporter gene (Figure 2) . The reporter genes are expressed from an IRES element placed 3’ of the gene of interest and both the reporter gene and the gene of interest are driven by the viral LTR promoter. The reporter genes include human placental alkaline phosphatase (PLAP; link to FieldsBerry et al. 1992), nuclear lacZ (NINII vector), and GFP (a vector acquired from Gary Nolan, Stanford University). For a discussion of the uses of the various reporter genes, see Chapter . Also, for a discussion of the use of the viral LTR promoter vs. internal promoters, see Gaiano et al. (1999).

CHICK REPLICATION-INCOMPETENT VECTORS. The chick retroviral vectors can replicate and transduce a nonviral gene of up to 2.5 Kb (Hughes et al. 1987). In contrast, the chick replication-incompetent vectors cannot replicate, but can transduce one or more nonviral genes. These vectors have been useful for lineage tracing and also encode a histochemical reporter gene and an oligonucleotide library (Golden et al. 1995). We have recently made a new generation of these vectors and have optimized their use for in vivo infections (Figure 3) . (link to Chen et al. 1999). They are most infectious if encapsidated in the VSV G protein, giving up to 350 fold more efficient infection in vivo relative to the normal viral envelope of the avian vectors (Figure 4) . Future vector projects include making these vectors more readily able to accommodate a large insert and a histochemical reporter gene, as well as utilize internal promoters.

Figure 1.

Figure 2. The murine retrovirus, LIA, is shown. A non-viral gene or transgene, is typically cloned into a site just downstream of the virus's normal start site for the gag gene. The transgenes uses its own ATG for translation. The Moloney Murine Leukemia Virus LTR promoter is used for transcription of both the transgene and the IRES-AP cassette. The IRES directs translation of the human placental alkaline phosphatase gene (PLAP), which enables histochemical detetion of infected cells. Titers of this vector are usually in the 10^7-10^8 CFU/ml following concentration. For examples of studies using this vector, see Bao and Cepko (1997) or Morrow et al. (1999). Alternative versions of this vector include GFP or nLacZ (NINII) in place of PLAP.

Figure 3. A series of avian retroviral replication-incompetent vectors have been created to enable expression of a transgene in chicks. The RISAP vector (shown in C and D) allows expression of a transgene and the human placental alkaline phosphatase gene, PLAP. PLAP enables histochemical detection of infected cells. The gag protein can also enable detection of infected cells via immuno-histochemistry using anti-gag antibodies. The transgene is expressed from a spliced mRNA, or a full length unspliced mRNA, via translation enabled by an IRES. PLAP is translated from a spliced transcript. All transcripts are driven by the Avian Leukosis Virus LTR promoter. The pSLIRES11 plasmid is used for convenient insertion of a transgene downstream of an IRES such that the transgene is in frame with the IRES' ATG. See Chen et al. (1999) for details.

Figure 4. The VSV G protein endows avian retroviral vectors with a high efficiency of infection in vivo. Comparable amounts of an avian replication-incompetent vector, RIA-AP, encoding PLAP were injected into developing chick embryos. RIA-AP (G) virion particles contained VSV G protein on their surface and RIA-AP (A) virions contained the avian retroviral A env protein on their surface. Embryos were injected at stage 18 into the eye (A and B), stage 10 into the neural tube (E and F), or stage 18 into the limb bud or heart regions (C, D, G, H). Embryos injected with RIA-AP (A) are shown in panels A, C, E., and those injected with RIA-AP (G) in panels B, F, D, and H. Sections of infected limbs are shown in G and H. Approximately 3 days postinfection, the embryos were stained to reveal PLAP activity; red arrowheads indicate the limb regions and blue arrowheads the heart.