mrna mrna mrna mrna GCC(A/G)CC
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1 - ( ) - * - :. * zomorodi@nigeb.ac.ir. :. (CMV) IX :. IX ( ) pcdna3 ( ) IX. (CHO). IX IX :.. IX :. IX IX : // : - // :
2 /.(-) ).( ' '.(-) mrna - () m7g.() mrna ( ) (AUG) ) () ( - ( ) mrna. mrna.( ) ' GCC(A/G)CC (AUG).( ) /.() ex vivo in vivo B. X /.()..().(-).()
3 / MWG. hfix-k-e1-f hfixe4-r : LB. ) Xgal (IPTG) ( NotI. (G-418) T4 DNA Ligase DraI PCR () BamHI.. ptz57rt. () DMEM. () Hams-F12. CHO :DNA DNA.() pet26-hfix-i ) K-E1-I1-E2-4 cdna ١ Luria-Bertani ٢ Dulbecco s modified Eagle s medium 5'GGATCCGCCACCATGCA GCGCGTGAACATGAT 3 5' CCT TGC AAC TGC CGCC ATTTA AAC 3'.().( ).(). (ATG) (). CHO : ( -). CHO pet26-. () hfix-i. PCR (-) pcdna3..
4 /.... : PTT. / /) (// /. µl µl PTT µl mm µl.. : pcdna3-k-hfix-i CHO. /. ( cdna. hfix-k-e1-f hfixe4-r hfix-k-e1-f. DraI BamHI : pcdna3-k-hfix-i -) ABI 373 (MWG. : CHO Hams-F12 CO2 % / DMEM 100 µg/ml G U/ml.. CHO / /. µl - µl ( ). µg. ml 3 Activated Partial Thromboplastine
5 / : BamHI/DraI PCR () pet26-hfix-i (K-E1-I1-E2-4)
6 /. pet26-k-hfix-i DraI XhoI BamHI.(- ) pet26-k-hfix-i DraI /BamHI DraI /BamHI ).(- /. ptz57r/t. ptz57r/t- K-E1-I1-E2-4 ptz57r/t- K-E1-I1-E2-4 () pet26-hfix K-E1-I1-E2-4 BamHI/DraI E1-E2-4 pet26-hfix. pet26-k-hfix-i. K-hFIX-I pcdna3-k-hfix-i. ( - ) : CHO :2 :. Hind III :pet26-k-hfix-i :. : pet26-k-hfix-i : pet26-k- hfix-i : pet26 pet26- : DraI BamH I K-hFIX-I-pET26 : DraI NotI BamHI : Hind III :.pcdna3-k-hfix-i :.NotI BamHI hfix-i : NotI pcdna3-k-hfix-i : BamHI pcdna3-k-hfix-i.noti BamHI pcdna3-k-hfix-i pcdna3 pet26-k-hfix-i cdna BamHI/NotI pcdna3- pcdna3
7 /. :. pcdna3-hfix-i pcdna3 IX.( ) CHO pcdna3-hfix- pcdna3-k-hfix-i () I. (pcdna3).. µg/ml.. pcdna3-k-hfix-i :. :. :.. NC pcdna3 N.
8 / s.() GCC(A/G)CC mrna.( ) - ).(. ( ) GCC(A/G)CC mrna -.() (GCC(A/G)CCAUG). mrna.() (A)..().(). cdna cdna /. IX. ).() ( ) IX. (..().(-) - mrna.( ) mrna - (AUG) mrna.(-) mrna.( ).()
9 / / / face.().. CMV CHO. - mrna - GCCACC. CHO. References: 1.Garlick JA, Fenjves ES. Keratinocyte gene transfer and gene therapy. Crit Rev Oral Biol Med 1996; 7: White SJ, Page SM, Margaritis P, et al. Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo. Hum Gene Therapy 1998; Makrides SC. Components of vectors for gene transfer and expression in mammalian cells. Protein Expr Purif 1999; 17: Fong YW, Zhou Q. Stimulatory effect of splicing factors on transcriptional elongation. Nature 2001; 414: Ismail SI, Kingsman SM, Kingsman, A J, et al. Split-Intron Retroviral Vectors: Enhanced Expression with Improved Safety. J Virol 2000; 74: Rohrer J, Conley ME. Transcriptional Regulatory Elements within the first intron of bruton s tyrosine kinase. Blood 1998; 91: Hirosawa S, Fahner J B, Salier JP, et al. Structural and functional basis of the developmental regulation of human coagulation factor IX gene: factor IX Leyden. Develop Biol 1990; 87: Larizza A, Makalowski W, Pesole G, et al. Evolutionary dynamics of mammalian mrna untranslated regions by comparative analysis of orthologous human, artiodactyl and rodent gene pairs. Comput Chem 2002; 26:
10 / 9. Pesole G, Mignone F, Gissi C, et al. Structural and functional features of eukaryotic mrna untranslated regions. Gene 2001; 276: Spicher A, Guicherit OM, Duret L, etal. Highly conserved RNA sequences that are sensors of environmental stress. Mol Cell Biol 1998; 18: IX Kozak M. An Analysis of Vertebrate mrna Sequences: Intimations of Translational Control. J Cell Biol 1991; 115: Shatkin AJ. Capping of eucaryotic mrnas. Cell 1976; 9 : Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 1986; 44 : Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987; 196: Kozak M. Context effects and (inefficient) initiation at non-aug codons in eucaryotic cell-free translation systems. Mol Cell Biol 1989; 9 : Kozak M. Translation of insulin-related polypeptides from messenger RNAs with tandemly reiterated copies of the ribosome binding site. Cell 1983; 34 : Kozak M. A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes. Gene Expression. 1991; 1 : Kozak M. Effects of long 5' leader sequences on initiation by eukaryotic ribosomes in vitro. Gene Expression 1991; 1: Kozak M. Influences of mrna secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci USA 1986; 83: Kozak M. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci / USA 1990; 87 : Kozak M. Regulation of protein synthesis in virusinfected animal cells. Adv Virus Res 1986; 31: Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position 4 but is not generally affected by the nucleotides in positions 5 and 6. EMBO J 1997; 16: Harkins S, Cornell CT, Whitton JL. Analysis of Translational Initiation in Coxsackievirus B3 Suggests an Alternative Explanation for the High Frequency of R+4 in the Eukaryotic Consensus Motif. J Virol 2005; 79: Michelon A, Michelon M, Simionatto S, et al. Effect of the kozak sequence on seroconversion of mice immunized with a DNA vaccine agenst swine colibacilosis. Braz J Microbiol 2003; 34: Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. USA: Cold spring harbor laboratory press, Miao CH, Ohashi K, Patijn GA, et al. Inclusion of the hepatic locus control region, an intron and untrranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Molecular Therapy 2000; 1: Caveneer, Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res 1987, 15: Johansen H, Schumperli D, Rosenberg M. Affecting gene expression by altering the length and sequence of the 5' leader. Proc Natl Acad Sci USA 1984; 81: Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin invivo. Nature 1984; 308: Kozak M. Selection of initiation sites by eukaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin. Nucleic Acids Res 1984; 12: Christopher R, Erwin M A, Helmrath C E, et al. Intestinal overexpression of EGF in transgenic mice enhances adaptation after small bowel resection. Am J Physiol Gastrointest Liver Physiol 1999; 277: Pang CP, Crossley M, Kent G, et al. Comparative sequence analysis of mammalian factor IX promoters. Nucleic Acids Research 1990; 18:
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