478 Development and Production of Marker Free Non Viral Vectors and archea Self splicing introns are mobile elements that in some cases invade the genome at an intronless allele (call ed retrohoming)[.]
and archea Self splicing introns are mobile elements that in some cases invade the genome at an intronless allele (call ed retrohoming) or at seemingly random places (called retrotransposition) Group II introns share a common structure that has bccn depicted as six domains form a pinwheel Specific sequences in the first domain are used by the intron to find its site in the target DNA for retrohoming The bacterial group II intron LI.LlrB can be retargeted to invade new places by adjustment of'the IBS sequences Furthermore, group II introns carry an open reading frame for a protein that assists the intron in its splicing and homing process This ORF can be provided separately, and thc space of the removed ORF can be filled with sequences of choice Our aim is to repair genes in mammalian cells by using the LI.Ltrb group II intron We approach this aim from different ang les First, we investigate how different types of cargo affect the splicing and homing efficiencies of the intron, Size and nature of the inserted sequence seem to affect intron homing efficiencies, but so far, the determining factor is not clear Second, we will introduce the group II intron to mitochondria, which, because they are evo lutionary related to bacteria, are expected to have the right environment for the intron to perform its splicing and homing reactions We will deliver DNA encoding the group II intron to mitochondria through bacterial conjugation where it will be expressed from a T7 promoter As cargo, we will introduce different sequences as part ofthe group II intron to repair mitochondrial diseases Third, we have introduced the L1.LtrB intron to mammalian 293-cells and will optimize splicing and homing efficiencies, the presence of both sequence abolished the individual effect In hypoxia, the presence ofHRE in uP augmented VEGF production to 200 %, meanwhile the presence of DTS diminished to 50 % and the presence of both sequence diminished even more (Figure I) In normoxia and in vivo , the presence ofI-IRE in the vector diminished drastically VEGF production (ca folds) , meanwhile the presence ofDTS or both did not affect in comparison to uP-VEGF On the other hand, in hypoxia, the presence of I-IRE augmented two fold ofVEGF in comparison to uP· VEGF and ten fold in comparison to normoxia Our results indicate that vectors constructed with DTS or HRE work well in normoxia or hypoxia, respectively, However, if those vectors are used in opposite condition for transfection, gene expression level is diminished significantly We hypothesize that the active transcription factors responsible for carrying a DNA sequence to nucleus are different in normoxia and hypoxia; consequently, those factors can facilitate or make difficult in transporting vectors with DTS or HRE u,o 12,0 10.0 8,0 6,0 1.0 0.0 477 Effects of Hypoxia Response Element and DNA Targeting Sequence in VEGF Gene Expression in Hypoxia and Normoxia Presence of transcription factor binding sites or nuclear localization signal in plasmid vectors facilitates transport ofplasm ids into the nucleus; consequently high level ofgene expression can be reached with fewer amounts of vectors A sequence of 72 bp located in an enhancer/early promoter sequence of Simian Virus 40 (DTS) is recognized by more then 10 tran scription factors in normoxic condition, which facilitate the transport ofa DNA with DTS Hypoxia response element (I-IRE) sequence is recognized by hypoxia-inducible factor (HIF), which is the main transcription factor recruited in hypoxia Transport of plasmid with HIF or DTS in normoxia and hypoxia is not clear and we report about it Methods-The plasmid vector uP-VEGF was designed to contain the sequence of CMV promotor/enhancer and the human VEGF gene Oligonucleotide sequences corresponding to HRE (1-1) and DTS (S) were inserted into the uP-VEGF plasmid to generate uPS-VEGF, uPH-VEGF and uPHS-VEGF In vitro gene expression was evaluated by ELISA in HEK293 cells transfected by calcium phosphate method In vitro hypoxic condition was created adding cobalt chloride in the culture medium For in vivo gene expression study, 100 ug ofeach plasmid was injected in the thigh of 10-12 weeks Balb/c mice After 48h , the crude extract from the muscles was used for quantification of VEGF by ELISA In vivo hypoxia condition was established by induction of acute limb ischemia Briefly, the femoral artery was excised from its origin ofthe external iliac artery to the distal point All branches of femoral artery were ligated Results and Discussion-In normoxia and in vitro, the presence of one copy of DTS or HRE in uP vector augmented 25 % of VEGF production, but S184 p 'Yax I,'E Gf t 6~ i \p ·VEC#'l65 =r .L I- = ~I=- I ~ no":::tt: f- · 2,0 uPH·VEGf'l6S l#l'S-~Gf'16!i ~ ~ H S-VEGf't65 D••heob O _t\oll'lC.N" 10.0 Chester B Sacramento,' Jane Z Moracs,' Rodrigo Tambcllini,' Sang W Han.' 'CINTERGEN UNIFESP, Sao Paulo Brazil: "Biophystca UNIFESP, Sao Paulo, Brazil: 'Nefrology; UNIF/!.-SP, Sao Paulo, Brazil r ' ,0 r ~ "' ;: ~ ~ :f \Mo\EVr~ - - - - ~ ~ P·W