Detection of deletion mutants by PCR. Primers annealing to MLV 4070A env and LTR U3 specific sequences were used to amplify integrated proviral vector DNA isolated from (A) in vitro infected NIH-3T3, HEK293, and U87-MG cells and (B) in vivo infected subcutaneous U87-MG tumours growing in Hsd:athymic Nude-Foxn1numice. DNA was isolated from (A) different infection cycles or (B) from infected mice, indicated with numbers at the top of each lane, respectively. Nomenclature of PCR fragments is derived from the vector name as follows: the two-letter prefixes AC-, AU-, and AK- denote mutants derived from vectors ACE-GFP, ACEU3-GFP, and Akv4070AeGFP or AkvB4070AeGFP, respectively. The subsequent letter or number denotes mutants emerging in NIH-3T3 (N), HEK293 (2), U87-MG (U) or solid tumours (T), respectively. The final digit indicates the order of appearance of the PCR-fragment. In the case of revertants composed of more than one deletion, numbers and letters describing the participating recombinations are shown. Vector mutants which appear to have arisen consecutively contain the name of the putative precursor vector mutant in parentheses. White arrows indicate the size of the PCR product resulting from amplification of the respective parental vector. N = DNA from uninfected cultured cells or tumour tissue. Empty lanes indicate either that DNA isolation was unsuccessful or that DNA was isolated from tumour tissue apparently not infected. M = molecular size marker. Agarose gel images from PCR performed on in vivo infected tumour material 20d post-infection amended from Journal of Virology, July 2007, p. 6973–6983, Vol. 81, No. 13, doi:10.1128/JVI.02470-06, with permission from American Society for Microbiology.