Eureka! Institutional Repository

Cervical Cancer is the fourth most common cancer and a major health problem worldwide. Cervical cancer has been associated with various pathological conditions with the major association with HPV infection. Actually, "high risk" types of HPV cause more than 70% of cervical cancers. However, HPV is not the only pathological condition who have been associated with cervical cancer. Considerable scientific research is based on the relationship between C. trachomatis , a common sexually transmitted disease, and cervical cancer. Interestingly, the studies have shown that C. trachomatis is one of the major co-factor in HPV induced cervical carcinogenesis. Thus, there is an interaction between Cervical cancer, HPV and C. trachomatis infection. A big concern in several studies was the behaviour of APOBECs gene, which is lead to mutations, 68% of them are in the bladder, cervical, breast, head and neck, and lung cancers. Previous Experiments based on transcriptomic analysis of C. trachomatis infected mouse END1E6/E7 and HPV negative primary human ectocervical cells, performed at Department of Molecular Biology from Max Planck Institute for infection biology were based on the role of Chlamydia infection in the development of cervical cancers. These experiments showed that the expression of APOBEC3A, APOBEC3B and APOBEC3G genes after Chlamydia trachomatis infection was upregulated at both RNA and DNA level (Chumduri, Gurumurthy et al, unpublished). The aim of this research project is a)Generate CRISPR/Cas9 mediated knockout cells lines of Apobec3B, Apobec3A, Apobec3G b)Confirm the Knockouts and c)Investigate the effects of knockouts during C. trachomatis infection. We successful cloned gRNA constructs in the lentivirus vectors which also consist the Cas9 expression cassette. We were also succed in generating the lentiviruses for three independent gRNAs targeting APOBEC3B and transduction of END1/E6E7 cells. However, subsequent analysis by Western Blotting revealed that we do not have knockouts.