A series of replication-competent, avian-specific retroviral vectors known as RCAS or RCAN have been developed by Hughes et al. (1) and used successfully by a rapidly expanding number of groups to assess gene function directly (e.g., refs. 2-14). These proviral vectors are derived from the Rous sarcoma virus and contain a unique ClaI restriction site in place of the region normally encoding the src oncogene, into which foreign DNA fragments of up to approx 2.4 kb can be inserted. An Escherichia coli plasmid backbone allows the gene of choice to be introduced by standard subcloning techniques, whereas retention of the viral long terminal repeat (LTR) sequences together with sequences encoding the viral gag, pol, and env genes facilitates viral replication and transmission. RCAN is a variant of RCAS from which the splice acceptor immediately upstream of the ClaI site has been removed preventing translation of the inserted gene and acts as a control for nonspecific effects owing to viral infection.
These proviral vectors can be efficiently transfected into cultured chick embryo fibroblasts (CEFs) using standard techniques, and supernatant containing infectious virions easily collected and concentrated to yield high-titer viral stocks, which can then be used to infect chick embryos of susceptible strains. Alternatively, transfected CEF cells can be grafted directly into host embryos. To increase their host range and to make it possible to introduce two vectors carrying different genes into the same cell, vectors containing different subgroup env genes have been constructed. The viral surface glycoprotein encoded by the env gene primarily determines the host range specificity of the virus, and vectors are currently available that contain env genes derived from subgroups A, B, D (ref. 1) as well as subgroup E (ref. 15).
From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ
The accessibility of the avian embryo coupled with its amenability to microsurgical manipulation means that expression of the retrovirally introduced gene in ovo can be easily manipulated simply by varying the site and time of infection and/or limited to a specific region by transplantation of infected tissue or CEF cells into a host not susceptible to infection. Specific examples of such manipulations are to be found in the literature cited herein. Detailed protocols for the numerous steps involved in the production of high-titer viral stocks and/or transfected CEF cells that can subsequently be used to assess gene function directly within the developing chick embryo are outlined below. A protocol for the grafting of transfected cell pellets is also included. Detailed information and protocols for injection of high-titer viral stocks can be found in an excellent review by Morgan and Fekete (16).
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