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Functional genome analysis using the retroviral integration sites permissive for provirus expression in human cells
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Španielová, Hana (referee)
The expression of retroviral genes depends on the establishment of the provirus - the DNA copy of retroviral genome integrated into the host genome. The transcriptional state of provirus is then influenced by the environment at the site of integration. The phenomenon of proviral silencing is an obstacle to the usage of retroviral vectors and a barrier to the eradication of human immunodeficiency virus type 1 (HIV-1) from infected individuals. Taking advantage of single cell clones bearing one provirus, this diploma thesis investigates the distribution of (epi)genomic features at the sites occupied by stably expressed proviruses. In total, long-term expression profiles of 245 and 255 clones carrying avian sarcoma-leucosis virus (ASLV) and HIV-1, respectively, were obtained. The database-based analysis of 42 integration sites of ASLV and three integration sites of HIV-1 proviruses shows that proviral stable expression highly correlates with the transcriptional start sites (TSS) at the sites of integration. Histone marks characteristic for the proximity of active TSSs and regulatory elements at the sites of integration of stably expressed proviruses confirm this finding. The results presented in this thesis could inspire other analyses investigating the relationship between the integration site and the...
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Mechanismy kontroly endogenních retrovirů v hostitelské buňce.
Matoušková, Magda ; Hejnar, Jiří (advisor) ; Mělková, Zora (referee) ; Pačes, Jan (referee)
In my thesis I am dealing with human endogenous retroviruses (HERVs), which are involved in placenta development, and with porcine endogenous retroviruses (PERVs) in the context of the risk of their transmission to a patient xenotransplanted with a pig organ. We have shown DNA methylation to be an important silencing mechanism regulating HERV as well as PERV. (1) Whereas in placenta the demethylation of HERVs ERVWE1 and ERVFRDE1 is crucial for its correct function, in the testis it is connected with seminoma development. (2) It seems that methylation is partially responsible for low PERV expression in tissues and notably reduces the risk of zoonotic transmission during xenotransplantation. (3) However, the risk remains because in contrast with mouse and rat cells, some human cells are permissive to porcine retroviruses in vitro thanks to functional receptors and their inability to efficiently silence the integrated PERVs.
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Genomic preferences in integration and expression of retroviruses and retrovirus-derived vectors
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Žíla, Vojtěch (referee)
Integration and provirus establishment are the key steps of retroviral life cycle. Genome-wide studies show that the integration is not a random process and that groups of related retroviruses display distinguishable patterns of integration preferences. The most rated genomic features forming the integration preferences of retroviruses are transcription units, transcription start sites and CpG islands. Whereas extending knowledge of genome structure, new, mainly epigenetic marks, which have a relationship to retroviral preferences are being defined. The integration into a specific genome region has a straight influence on the provirus expression and therefore on the production of virus progeny. While integration into some regions results in provirus silencing which is managed and maintained by variety of factors, some other genomic regions are vice-versa capable of stable provirus expression maintenance. These findings have implications for construction of safe and efficient retroviral vectors as well as for use of modified retroviruses as markers for an epigenetic and expression profile determination of genome regions.
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Genová imunoterapie nádoru: DNA vakcíny proti HPV16
Poláková, Ingrid ; Šmahel, Michal (advisor) ; Hejnar, Jiří (referee) ; Reiniš, Milan (referee)
of PhD. Thesis Gene Immunotherapy of Cancer: DNA Vaccines against HPV 16 Mgr. Ingrid Poláková Cervical carcinoma (CC) represents the second most frequent cancer in women, mostly associated with human papillomavirus (HPV) infection. Nowadays, two prophylactic vaccines, protecting against HPV 16 and HPV 18, are licensed. Nevertheless, development of therapeutic vaccines is desirable to eliminate current HPV infections and to treat progressing tumours. Suitable targets for vaccination are viral E6 and E7 oncoproteins. Since its discovery, DNA vaccination has become an effective strategy for development of vaccines against cancer including CC. Unfortunately, the immunogenicity of DNA vaccines in large animals and particularly in humans is low. Therefore, several ongoing studies are focused on strategies enhancing the efficacy and safety of DNA vaccines. In this work, the immunogenicity of DNA vaccines against HPV 16 delivered by a gene gun was evaluated after the fusion of the E7 and E6 genes with GUS. The increased steady-state level of the E7GGG.GUS deletion mutants and the GUS.E7GGG fusion protein enhanced the production of E7-specific antibodies after immunisation with these vaccines but did not improve the CTL response. Joining of the signal sequence with GUS.E7GGG led to ER-localisation of the...
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Alterations of the oncogenic properties of tumor cell lines by modulating oncogene expression. Example of v-src transformed chicken cell lines
Kovářová, Denisa ; Hejnar, Jiří (advisor) ; Španielová, Hana (referee)
4 Alterations of the oncogenic properties of tumor cell lines by modulating oncogene expression. Example of v-src transformed chicken cell lines Abstract v-Src protein tyrosine kinase is the product of the transforming gene transduced by avian Rous sarcoma virus. In contrast to proto-oncogene c-src, v-src lacks the negative-regulatory C-terminal domain and consequently shows a higher level of activity and transforming ability. In addition, v-Src contains point mutations throughout its coding region that probably contribute to the high level of intrinsic kinase activity. Long terminal repeats (LTR) comprise strong promoter-enhancer sequences and ensure efficient expression of the v-src gene. v-Src protein has a strong transforming potential in vitro and induces tumor development and growth in vivo. Moreover, it is implicated in metastatic formation. In several cancer types the elevated c-Src kinase activity caused pleiotropic cellular responses inducing transformation and metastasis. The aim of this diploma work was to reveal the role of v-Src in mediating tumor and metastatic progression in chicken cell lines PR9692 and PR9692-E9. Despite the low propensity of the chicken cells to immortalization, comparatively high immoratlization efficiency was observed in cells from ex vivo tumours growing progressively...
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Improvement of retroviral vectors for efficient gene transfer
Šenigl, Filip ; Hejnar, Jiří (advisor) ; Šmahel, Michal (referee) ; Ruml, Tomáš (referee)
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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