1. The choice of viral subgroup should also be considered. Although subgroup A retroviral vectors have been routinely used for limb studies (2,3,5-7,12-14), subgroup B vectors have been found to infect developing neural tissue more efficiently (20).

2. Before starting the cloning procedure, it is also important to consider how expression of the inserted gene will be determined. If antibodies are not available with which to detect the encoded protein, it may be appropriate to label the protein with an expression tag, such as Myc (21) or FLAG (22), provided such a tag does not interfere with protein function. However, if such expression tags are used, it is important first to ensure that the antibody against the tag does not crossreact with endogenous chick proteins.

3. Where possible, expression of the encoded protein should be assayed immunohistochemically via Western blots of total protein extracts and/or supernatant (if secreted) from transfected CEF cells. Protein function should also be assayed if possible. Alternatively, Northern blot analysis of mRNA from trans-fected CEF cells could be used to determine if the inserted sequences are being properly spliced to produce the transcript required for translation of the exogenous gene. Three alternatively spliced mRNA viral transcripts should be produced in transfected cells. It is the smallest splice variant, whose abundance should be >10% of the total viral RNA, which is used for translation of the exogenous gene. In addition, the pSLAX12 adapter plasmid containing the inserted sequences could be used in an in vitro transcription/translation assay to determine if protein of the correct size can be translated. Kits are readily available from several manufacturers. We routinely use the TNT-coupled wheat germ extract system (L4120) and tRNAnscend nonradioactive translation detection system (L5080) from Promega.

4. The lack of endogenous retroviruses makes line 0 CEFs desirable for growing viral stocks, since the possibility of recombination between the exogenous virus being introduced and endogenous viruses is minimized and viral spread can be readily determined by immunostaining using the monoclonal 3C2 anti-GAG antibody (18).

5. CEF cells are very sensitive to the type of plasticware used. In our experience, they preferentially adhere to and grow best on tissue-culture dishes supplied by Griener, Corning, and/or Nunc.

6. To introduce two vectors carrying different genes into the same CEF cells, separate transfections (using vectors containing different subgroup env genes) should be done sequentially. CEF cells should be passaged two to three times between transfections.

Fig. 1. Immunohistochemical analysis using the 3C2 anti-GAG monoclonal antibody

(18) of (A) uninfected and (B) RCASBP(A)-infected CEF cells (courtesy of I. Campbell).



Fig. 1. Immunohistochemical analysis using the 3C2 anti-GAG monoclonal antibody

(18) of (A) uninfected and (B) RCASBP(A)-infected CEF cells (courtesy of I. Campbell).

7. The titers obtained after 4-6 h of incubation are almost as high as those obtained after 16 h (i.e., O/N). Therefore, multiple collections of supernatant could also be done at 4-6 h intervals.

8. Concentrated viral stocks can maintain their titer for months and/or years when stored at -70°C.

9. The titer of concentrated viral stocks varies considerably, depending on the virus, confluence, and fitness of the cells used for collection and the volume of supernatant used to resuspend the viral pellet. In general, titers below 108 infectious virions/mL do not produce high-efficiency infection and would thus not be considered useful.

10. The cell pellet should be compact. If it is too dispersed, the cells cannot be used for grafting, since the cell pellet will disintegrate during transfer and/or subsequent manipulations.

11. Plastic disposable pipets should not be used to transfer the cell pellet, since we have found that the pellet often sticks to the side of the pipet.

12. When adding the pellet to the embryo, it is very important to watch where the pellet goes, since it is often invisible on the embryo.

13. Manipulations, such as the grafting of cell pellets, can sometimes delay development.

14. Although compact cell pellets can be implanted into a specific region of the embryo, cells often disperse within 24-48 h (e.g., see ref. 3).

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