1. Sequence analysis programs MacVector (Macintosh platform) or DSGene (PC platform) can be purchased from Accelrys (www.accelrys.com). Analysis software is necessary in order to design PCR primers for the protein moiety, as well as to verify cloned sequences generated later in the protocol.
2. Caution: EtBr is a potent mutagen. When preparing EtBr solution, wear gloves, a dust mask, and eyewear to protect against particulate matter. Store solution in amber glass labeled with a clear warning. Always wear gloves when dispensing EtBr solution and handling gels to which EtBr has been added. Dispose of contaminated tips and used gels in accordance with institutional guidelines for the handling of mutagenic materials.
3. Depending on the application, the experimenter may choose the protein moiety to be genetically fused to murine Fc. However, any putative transmembrane regions must be excluded, as their inclusion will prevent soluble expression of the fusion protein by NS0 cells. It is reasonable to assume that a molecular weight limit exists, above which NS0 cells may yield insufficient amounts of recombinant protein.
4. The primer templates given were designed for the user's convenience, and represent the templates used in our laboratory in most cases. However, if the target contains either of the pertinent RE sites listed, then the user must redesign her target primers to replace that RE site with one that (1) appears in the multiple cloning site of the pEE12 expression vector, and (2) does not appear in the target sequence. The pEE12 vector map is obtainable from Lonza Biologics.
5. In the absence of the target cDNA template, no PCR product is expected. If the negative control tube yields PCR product, this suggests DNA contamination, and common reagents (dNTP, buffer, dH2O, vent) should be replaced. If possible, assemble a positive control PCR tube, substituting unrelated cDNA and PCR primers known to yield product. This reaction will validate the dNTP, buffer, dH2O, and Vent polymerase used.
6. Tube 3 should contain clones bearing the pEE12/muFc master vector, and thus should yield the most colonies. Tube 2 ("vector only") indicates the likelihood that the pEE12 vector was ligated to itself without inclusion of the muFc insert. If the number of colonies yielded by tube 2 approximates tube 3, then the likelihood of successful ligation is low, and litigation should be repeated. If subsequent tube 2 reaction yield numerous colonies, consider using calf intestinal alkaline phos-phatase (CIP) to interfere with vector self-litigation (according to manufacturer's instruction). Tube 1 ("no ligase") should yield no colonies, though in practice may yield <10 clones; this tube indicates the extent to which pEE12 was successfully digested by HindlII and £coRI. If tube 1 yields more than 10 colonies, the user should redigest vector and insert (Subheading 3.2.1.).
7. If necessary, less than 40 pg of transfection vector may be used to perform the stable transfection, but the number of NS0 cells and 96-well plates must be adjusted accordingly.
8. The maximum volume of the cuvet is 1 mL, which must contain the volume of transfection vector digest; NEB cat. no. M 0290S). 1 X 107 total NS0 cells. If the volume of the digest varies from the total 100 pL volume, adjust the volume of NS0 cells accordingly.
9. It is very important to not disturb the 96-well plates during this 3-4 wk incubation period. When the plates need to be checked for colonies, designate one plate that will be removed from the incubator to be checked from time to time.
10. For the Fc-specific ELISA, an appropriate positive control would be any murine Ab that is isotype IgG2a. Titer the positive control Ab so that an estimate of the user's Fc fusion protein can be made by comparing to known amounts of the positive control. For the negative control wells, add 50 pL of blocking buffer instead of 50 pL of transfectant medium.
11. In case the one cell/well subcloning is not successful, repeat subcloning at a concentration of two cells/well in an additional 96-well plate.
12. For protein moiety-specific ELISA, the appropriate positive control is the protein of interest. Only a small amount is needed (1-2 p,g), and may be available from commercial sources or another laboratory. As in Fc-specific ELISA, blocking buffer is used in negative control wells.
13. One can make adjustments to the serial dilution factor used in the ELISA. According to the OD values obtained in the initial Fc-specific ELISA, serial dilutions may be adjusted to 1:4, 1:5, or 1:10. An optimal range of dilutions enables the user to best compare the expression of individual subclones.
14. Ensure that the protein A column is continually kept moist with buffer. If the column is left to dry out, it may affect the yield of purified Fc fusion protein obtained.
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