Zdikonalování etrovirových vektorů pro účinný přenos genů

Šenigl, Filip

44 CONCLUSIONS Retroviral vectors are transcriptionally unstable in mammalian cells. The ASLV- derived vectors are the most affected by silencing. We modified the vector by insertion of the CpG island inner element (IE) into the vector LTR in three different positions in either sense or antisense orientation. Each vector variant exhibited a certain extent of stabilization. The vector with insertion of a tandem of two IEs between the enhancer and the promoter was the most stable and exhibited almost no silencing after four months of cultivation in rodent and human cells. The IE comprises two high-affinity Sp1 binding sites. The presence of Sp1 binding sites is important for the protective effect of IE, but at least a part of the entire anti-silencing capacity is maintained in IE with mutated Sp1 sites. We identified the Tvc receptor of ASLV. The tvc gene encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. To confirm the identification of the Tvc receptor, we disrupted both tvc alleles in normally susceptible DT40 cells. The DT40 tvc-/- clone was resistant to the ASLV(C) infection. We identified the mutation that results in decreased susceptibility to infection by ASLV subgroups B and D and resistance to ASLV subgroup E of line M...

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