REKOMBİNANT DNA TEKNOLOJİSİ III

1. REKOMBİNANT DNA TEKNOLOJİSİ III Doç.Dr.Öztürk ÖZDEMİR 2004-2005

2. The Tools of Molecular Biology

3. REKOMBİNASYON Rekombinasyon: Yenibileşim - yenidenoluşum. Bir molekülün-hücrenin, atasal “wild type” yada ilkin(orijinal) yapısından farklılık göstermesi durumudur. I - In vivo rekombinasyon II- In vitro rekombinasyon

4. Klon (Clone); Bir tek atasal diploid hücreden mitoz bölünme yoluyla birden fazla hücre eldesine denir. Rekombinant DNA teknolojisi ile sentezlenen identik DNA/gen kopyalarına denir. ”Gene cloning”

5. Genetik Klonlamada TarihçeGelişme Araştırıcı Yıl  Deniz hayvanlarında döllenme O.Hertwig 1875  İlk kez anne rahmi dışında döllenme L.Schenk 1878  Tüp ortamında insan yumurta hücresi döllendi MF Menkin 1944  Dondurulmuş sperm ile inek yumurtası döllendi 1952  Deney tüpünde döllenen bir memeli yavru doğdu 1959  Dondurulmuş embriyodan yavru fareler elde edildi 1972  Louise Brown isimli bebek deney tüpünde döllendi anne rahmine yerleştirilerek sağlıklı doğum yaptırıldı 1978  Avusturalyada donmuş embriyodan sağlıklı bir kız çocuğu elde edildi 1984

6. Genetik Klonlamada TarihçeGelişme Yıl  Kiralık anne Mary Beth bebeğini vermeyi redetti. 1986  Embriyo hücrelerinin çoğaltılmasıyla çok sayıda kuzu elde edildi 1987  İnsan embriyosu klonlandı çok tepki aldı J.Hall 1993  Dolly klonlandı I.Wilmut 1997  Fransada bir dananın 63 klonu elde edildi 1999  Totipotent stem hücrelerinden deneysel organogenezis 2000  Farede gen klonlama yöntemiyle insan kulağı gelişimi sağlandı 2001  Avusturalyada bir at klonlama ile Coada eşek doğurdu 2002  Amerikada ex vivo yapay rahim geliştirildi 2002

7. Klonlama Tipleri  DNA /Gen düzeyinde klonlama  Hücre düzeyinde klonlama  Organizma /çekirdek düzeyinde klonlama

8. Başarılı Klonlama Yapabilmek İçin Gen; Bağımsız olarak replike olabilmeli Konak hücreye kolaylıkla transfer edilebilmeli Seleksiyona olanak tanımalı

9. Memeli Hücrelerine Gen Transfer Teknikleri Microinjection***DAAE-Dextran MediatedElectroporationLipofection Calcium Phosphate*** Protoplast Fusion Polyprene Viral infection*** (Lentivirus, Retrovirus, Adenovirus) *: Yaygın kullanılan yöntemler

10. Genetik Klonlamada Kullanılan Vektörler  Plazmid 5-10 kb  Bakteriyofaj 20 kb  Cosmid 50 kb  YAC 100 kb  Baculovirus 150 kb  BAC 200 kb  PAC 250-300 kb  Lentivirus ** ~ 100-200 kb

11. The construction of Mammalian Transfection Vector For Expression of Cytosine- 5 Specific DNA Methyltransferase Gene M.Msp1 In Cultured CellsOzturk OZDEMIRReceived 01.05.1997

17. STOPLAZMİK KALITIM(YUMURTA HÜCRESİ) • Regülatör - modülatör proteinler • Yumurta polarity genler • Segmentasyondan sorumlu genler (25 adet) • Yumurta hücresindeAnterioposterior gradiyent farkı • Remodelling faktörler ; • zigotik effect • integrinler • transkripsiyon faktörleri • pair-rule genler • segment polarity genler • Homeodomeik, Hox (Homeobox) fetusa ait genler

18. Nükleer Transplantasyon • Wilmut ve arkadaşları donör hücre olarak 6 yaşında sağlıklı bir koyunun meme epitel hücresi ve resipient hücre olarak ise aynı koyunun metafaz II evresinde bekletilmiş enucleated yumurta hücresi kullandılar. Klonlama sonrası elde edilen ve annesiyle %100 aynı genotip ve fenotipte olan sağlıklı kuzuya DOLLY adını verdiler.

20. DOLLYDoç.Dr.Öztürk ÖZDEMİR

21. Nükleer KlonlamanınÖnemi  Yumurta hücresinin embriyogeneziste spermden farklı artı(+) öneminin olduğu,  Ökaryotik hücrenin G0 evresinde totipotent kromatin organizasyonu kazandıği,  Metafaz II evresinde yumurta hücresinin klonlama için en uygun stage olduğu,  Memelilerde eşeysiz üremenin mümkün olduğu,  Bir gen yerine çekirdeğin tamamının transplante olabileceği gösterildi

22. KlonlamaSonrasında;  Unipotent hücrenin totipotent hücreye dönüştürülmesi,  Sinir hücrelerinin rejenerasyonu,  Telomerlerde ” end replicatin problem”giderilerek, yaşlanmanın geciktirilmesi,  Stem hücrelerinden spesifik doku eldesi,  Epigenetik modifikasyonu ile kanser tedavisine yeni bir yaklaşım, “Ex vivo gene replacement” ile genetik tedavi ve  İnsan genom projesi önemli bir ivme kazanmıştır.

24. Goals of DNA Technology • Isolation of a particular gene or sequence • Production of large quantities of a gene product • Protein or RNA • Increased production efficiency for commercially made enzymes and drugs • Modification/improvement of existing organisms • Correction of genetic defects

25. Amplifying DNA Often we need large quantities of a particular DNA molecule or fragment for analysis. Two ways to do this:- 1. Insert DNA mol. in a plasmid and let it replicate in host >>> many identical copies (= ‘DNA cloning’) 2. Use PCR technique - automated multiple rounds of replication >>> many identical copies.

26. DNA Cloning • Purpose:- to amplify (bulk up) a small amount of DNA by inserting it into in a fast growing cell e.g. bacterium, so as bacterium divides we will have many copies of our DNA • 1. Obtain a DNA vector which can replicate inside a bacterial cell (plasmid or virus) which • 2. Insert DNA into vector - use restrictionenzyme • 3. Transform host cellsi.e. insert vector into host cell (e.g. bacterium) • 4. Clone host cells (along with desired DNA) • 5. Identify clones carrying DNA of interest

27. Vectors are convenient carriers of DNA. They are often viruses or plasmids. Usually are small circular DNA molecules and must be capable of replicating in the host cell The DNA of interest must be inserted into the vector.

28. Restriction Enzymes Cuts here Target or recognition sequence Restriction enzymes (R.E.) recognise target sequences and cut DNA in a specific manner. This R.E. leaves TTAA single stranded ends (‘sticky ends’) If you cut DNA of interest and plasmid with same restriction enzyme then you will have fragments with identical sticky ends.

29. AATT TTAA Recombinant plasmid Sticky ends will readily rejoin - so its possible to join 2 DNA’s from different sources Plasmids are usually chosen to have only one target site. DNA of interest can then insert into this site

30. Transformation of host and selection of desired clones • Bacteria are made to take up the recombinant plasmid & grown (cloned) in large numbers (TRANSFORMATION) • Bacteria carrying desired sequence can be selected. • Large amounts of DNA or proteins can be extracted

31. work with gene work with protein

32. Making a Genomic Library Genomic library = a complete collection of DNA fragments representing an organism’s entire genome. 1. Cut up genome into thousands of fragments with an R.E. 3. Result - a collection of bacterial colonies (clones) carrying all the foreign DNA fragments i.e. a genomic library 2. Insert each of these into separate plasmids and then into separate host cells.

33. A question for you - how will a cDNA library differ from a genomic library ? • Which would have more genes ? • What would be present in the clones in each case? • Promoters ? • Enhancers • Introns ? • Poly-T (from poly-A tail)?

34. long DNA short DNA Standards of known M.W. How do we identify DNA mols. of different sizes ? Gel Electrophoresis Run DNA fragments through a gel under influence of an electric current. Each of the DNA fragments travels through the gel at a constant speed appropriate for its size. Longer molecules move more slowly so don’t travel as far. See Fig 20.8

35. Polymerase Chain Reaction (PCR) Small amount of DNA can be amplified greatly - automated process involves:- • A DNA polymerase which is stable at high temperatures • specific primers to start off replication at known position. • Three step cycle: • Heat to separate DNA strands = Denaturation • Cool and allow primers to bind (Annealing) • Polymerize new DNA strands (Extension) Repeat steps 25 – 35 times >>> millions of copies of original DNA

36. Polymerase chain reaction Denaturation (95C) annealing (50C) Primer 15

37. Extension (72C) Polymerase chain reaction

38. Polymerase chain reaction Denature 13

39. Polymerase chain reaction Denature Anneal primers

40. Polymerase chain reaction Denature Anneal primers Extend

41. Polymerase chain reaction Denature

42. Polymerase chain reaction Denature Anneal primers

43. Polymerase chain reaction Denature Anneal primers Extend

44. Polymerase chain reaction Anneal primers Denature Extend

45. Bacterial Plasmids • Plasmids are small, circular DNA molecules in bacteria. • By inserting genes into plasmids, scientists can combine eukaryotic and prokaryotic DNA. (Recombinant DNA) • Bacterial cells continually replicate the foreign gene along with their DNA. • Cloning using plasmids can be used to: • Identify a particular protein a gene makes (ie: for study) • Produce large amounts of a particular protein/gene (ie: for use in medicine)

46. Restriction Enzymes • Also used to make recombinant DNA. • Specifically cut DNA molecules at precise base locations. (restriction)

47. Making Recombinant DNA (Fig 20.3) Making Recombinant DNA (Fig 20.3)

48. …Still Making Recombinant DNA

49. …Almost Recombinant

50. Why Use Bacteria as vectors? • Plasmids are easy to use to manipulate which genes are expressed in clones. 2. Bacteria replicate very quickly and allow you to produce a large number of a desired gene.