Before the development of this technology, analysis of tumor formation in mice with inactivating mutations was limited to mice heterozygous for the specific mutation [74] or chimeric mice partially derived from embryonic stem cells homozygous for a particular mutation [75].
Many studies based on single amino acid mutations or on chimeric antibodies have reported the involvement of the sequence E233LLGGP238 of IgG1 in the recognition of all FcγR (42), and especially residues Leu234 and Leu235 for FcγRI (37).
The use of PCR fusion combined with Gateway cloning (Life Technologies) enables rapid construction of vectors that drive expression of proteins with point mutations, insertions, deletions, or chimeric sequences without added recombination or restriction sites (Atanassov et al., 2009).
This may be ensured by the generation of large mutations (particularly deletions), numerous mutations with epistatic interactions, or chimeric viruses in which the structural proteins of a well-attenuated virus are replaced with those of the virus targeted for immunization.
In addition, point mutations within the JFH-1 cORF+1/ARFP orF or chimeric H77/JFH-1 strains do not affect virus production [16], [23].
The OID50 value for LR-GFP-226V with both the E2-D60G and E2-T211I mutations was indistinguishable from the OID50 values of 41855-GFP-226V or chimeric virus LR/41855-GFP-226V (p>0.1).
However, consensus or chimeric VLP approaches that require only the mutation of certain epitopes may provide a system that is more amenable for rapid production of vaccines that can adapt to emerging strains.
Importantly, and in strong contrast to what we found with the full-length RNA constructs in tamarins, the two 3'NTR mutations had no effect on the replication competence of either the parental or chimeric subgenomic GBV-B replicon RNAs (compare neo-RepDm3' and neo-RepD, as well as GBneoD/IIIHCm3' and GBneoD/IIIHC, Fig. 5B).
We isolated chimeric gC-GDNF or chimeric gC-BDNF constructs, and the resulting proteins were incorporated into HSV-1 virus particles.
This strategy using cloned DNA-derived wild type rather than attenuated DEN-4 has been previously used to investigate the genetic mutations of chimeric DEN-4 after Vero cell adaptation [21].
