Retroviral Delivery of ECM Genes to Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 197

sary to screen many colonies to find a clone containing a correct insert. Usually,
smaller colonies had a higher probability of maintaining a desirable insert. Other
E. coli strains, which are deficient in recombination, UV repair and SOS repair
(SURE cells) may be used in order to stabilize the insert.

4. For the Tet-off system, Tc or Dox needs to be maintained throughout transfection

and infection. The presence of Tc does not interfere with transfection or infec-
tion. In order to investigate gene regulation by Tc , infected cells can be split into
two plates and maintained in the absence and presence of Tc .

5. Virus containing supernatant can be frozen at -80 C for later infection, although

viral titer is decreased to 50% when the virus is frozen and thawed.

°

6. Retroviral titers vary widely depending upon different retroviral vector and pack-

aging cells. In general, constitutive vectors are likely to yield a high-titer virus
(10 6 infectious particles/mL) in comparison to a self-inactivating or a Tc-regulat-
able vector (10 4 infectious particles/mL). In order to avoid multiple infections,
which increase the number of integration events per cell, transduction is usually
performed at the multiplicity of infection of 0.1 (for example, 10 4 infectious
particles per 10 5 cells). This condition is likely to yield one viral integration
per genome.

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