EFFECTS OF SEPERATE OR SIMULTANEOUS INJECTION OF TWO DIFFERENT GENES (ENHANCED GREEN-FLUORESCENCE PROTEIN GENE, HUMAN GAMMA INTERFERON GENE)

Year 2009, Volume: 18 Issue: 2, 43 - 52, 01.06.2009

Korhan Arslan Haydar Bağış Munis Dündar

Abstract

hIFN-γ and EGFP gene constructs were separatelyor simultaneously injected to mouse embryos atpronuclear phase. Micro-injection were classifiedin three different experiment groups according toalone or simultaneous transfer of gen constructs. Infirst group co-injection of hIFN-γ+EGFP transgenes were performed. As a result of transfers,totally 39 offsprings were born, In molecularanalysis performed on DNAs obtained from thoseoffsprings, two were EGFP transgenic and one washIFN-γ transgenic. In second group micro-injectionof hIFN-γ trans-gene was performed. As a result oftransfers, totally 21 offsprings were born. Inmolecular analysis performed on DNAs obtainedfrom those offsprings totally two neonates werehIFN-γ transgenic. In third group micro-injection ofEGFP trans-gene was performed. As a result oftransfers, totally five offsprings were born, it wasfound that there were no transgenic offspring.Results presented in present study found thattransgenicity percentage of offsprings born as aresult of simultaneous micro-injection of twodifferent genes was high. If the protocol presentedin this study is applied to transgenic mice recoverystudies, it is possible to recover transgenic micecarrying two different genes separate. Thus, it willbe possible to save money and time

Keywords

Transgen, mikroenjeksiyon, EGFP, hIFN-γ, Ko-enjeksiyon

İki Farklı Genin (Güçlendirilmiş Yeşil Floresan Protein Geni, İnsan Gamma İnterferon Geni) Ayrı Ayrı veya Birlikte Mikroenjeksiyonunun Transgenik Fare Eldesi Üzerine Etkilerinin Araştırılması

Abstract

Sunulan çalışmada hIFN-γ ve EGFP genkonstraktlarının farklı konsantrasyonları birlikte veayrı ayrı pronüklear safhadaki fare embriyolarınamikroenjekte edilmiştir. Mikroenjeksiyon deneyleriaktarılan gen konstraktlarının yalnız ve/veya birlikteaktarımlarına göre üç farklı deney grubu çalışılmıştır.I.Deney Grubunda hIFN-γ ve EGFP genleri birliktemikroenjekte edilmiş, aktarımlar sonucunda toplam 39yavru doğmuştur. Yapılan moleküler analizlerdetoplam üç yavrunun transgenik olduğu belirlenmiştir.Üç transgenik yavrudan iki’si EGFP transgenik, birtanesi ise hIFN-γ transgeniktir. II. Deney Grubundayalnız hIFN-γ geni mikroenjekte edilmiş, aktarımlarsonucunda toplam 21 yavru doğmuştur. Yapılanmoleküler analizlerde toplam iki yavrunun hIFN-γtransgenik olduğu belirlenmiştir. III.Deney Grubundayalnız EGFP geni mikroenjekte edilmiş, aktarımlarsonucunda yavrulardan elde edilen DNA’lar ile yapılan moleküleranalizlerde Sunulan çalışma da elde edilen sonuçlar ile iki farklıgenin birlikte mikroenjeksiyonuyavruların transgenik olma yüzdelerinin yüksek olduğuortaya konmuştur. Sunulan çalışmada geliştirilenprotokolün transgenik fare üretim çalışmalarındauygulanması durumunda; iki farklı geni ayrı ayrı veyabirlikte edilebilecek, böylece ekonomi ve zaman açısındantasarruf sağlanabilecektir

Keywords

Transgene, Micro-injection, EGFP,

References

• Lo D, Burkley L, Flavell R, et al. Diabetes in transgenic mice expressing class II major histocompatibility complex molecules on beta cells. Cell 1988; 53: 159-168.
• Palmiter RD, Brinster RL, Hammer RE, et al. Dramatic growth of mice that develop from eggs microinjected with metallothionein- growth hormone fusion genes. Nature 1982; 300: 611–615.
• Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci 1980; 77: 7380- 7384.
• Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. Factors affecting the efeciency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci 1985; 82: 4438-4442.
• Jaenisch R. Transgenic animals. Science 1988; 240:1468–1474.
• Wheeler MB, Walters EM, Clark SG. Transgenic animals in biomedicine and agriculture: outlook for the future. Anim Reprod Sci 2003; 79: 265-289.
• Ekici A, Timur M, Bağış H. Ege Üniversitesi Su Ürünleri Dergisi 2006; 23. 211-214.
• Gordon JW, Ruddle FH. Integration and stable germ line transformation of genes injected into mouse pronuclei. Science 1981; 214: 1244- 1246.
• Janenisch R, Beatrice M. Simian virus 40 DNA sequences in DNA of healty adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl Acad Sci 1974; 71: 1250 -1254.
• Old RW, Primrose SB. Principles of gene manuplation an introduction to genetic engineering (3rd ed). Blackwell Scientific Publication 1985; pp 88-96.
• Jaenisch R, Dausman J, Cox V, Fan H. Infection of developing mouse embryos with murine leukemia virus: tissue specificity and genetic transmission of the virus. Hamatol Bluttransfus 1976; 19: 341-356.
• Wagner EF, Stewart TA, Mintz B. The human globin gene and a functional thymidine kinase gene in developing mice. Proc Natl Acad Sci 1981; 78: 5016–5020.
• Wagner TE, Hoppe PC, Jollick JD, Scholl DR, Hodinka RL, Gault JB. Microinjection of a rabbit globin gene in zygotes and its subsequent expression in adult mice and their offspring. Proc Natl Acad Sci 1981; 78: 6376– 6380.
• Jaenisch R. Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc Natl Acad Sci 1976; 73: 1260–1264.
• Hofker M, Deursen JV. Transgenic mouse methods and protocols. Humana Press, New Jersey 2002; pp 51-56.
• Auerbach AB. Production of functional transgenic microinjection. Acta Biochim Pol 2004; 51: 9- 31. DNA pronuclear
• Li S, Rosen JM. Glucocorticoid regulation of rat whey acidic protein gene expression involves hormone induced alterations of chromatin structure in the distal promoter region. Mol Endocrinol 1994; 8: 1328–1335.
• Chen TL, Lin YL, Lee YL, Yang NS, Chan MT. Expression of bioactive human interferon- gamma in transgenic rice cell suspension cultures. Transgenic Res 2004; 13: 499–510.
• Diamond MS, Harris E. Interferon inhibits dengue translation of viral RNA through a pkr- independent mechanism. Virology 2001; 25: 297-311. by preventing
• Lee CI, Kohn DB, Ekert JE, Tarantal AF. Morphological transduction of fetal monkey bone marrow- derived mesenchymal stem cell. Mol Ther 2004; 9: 112–123. and lentiviral
• Bagis H, Odaman H, Sagirkaya H, Dinnyes A. Production of transgenic mice from vitrified pronuclear-stage embryos. Mol Reprod Dev 2002; 61: 173–179.
• Bagis H, Odaman H, Dinnyes A. Exposure to warmer post-operative temperatures reduces hypothermia caused by anaesthesia and significantly increases the implantation rate of transferred embryos in mouse. Lab Anim 2004; 38: 50–54.
• Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the Mouse Embryo (2nd ed). Cold Spring Harbor Laboratory Press, Newyork 1994; pp 217-252.
• Zhang P, Hayat M, Joyce C, et al. Gene trans- fer, expression and inheritance of pRSV- rainbow trout GHcDNA in the common carp, cyprinus carpio. Mol Reproduc Dev 1990; 25: 13-25.
• Yamamura K, Kudo A, Ebihara T, et al. Cell- type-specific and regulated expression of a human heavy-chain immunoglobulin gene in transgenic mice (lipopolysaccharide induction/ B lymphocytes. Proc Natl Acad Sci 1986; 83: 2152-2156.
• Deborah OC, Borowski AH, Leung JD, et al. Generation of transgenic mice and germline transmission chromosome introduced into embryos by pronuclear microinjection. Chromosome Res 2000; 8: 181-191. artifcial
• Hogan B, Beddington R Constantini F, Lacy E. Manipulating Laboratory Manual (1st ed). Cold Spring Harbour Laboratory, Newyork 1986; pp 320- 331. Embryo: A