The Code For Life

1. Organism . Organ System Cell Nucleus Tissues The Code For Life

2. Chromosome Nucleus Genes The Code For Life Big nose Brown eyes Straight hair

3. Structural Biology Medicine and Biology at the Atomic Scale Organ  Tissue  Cell  Molecule  Atoms • A cell is an organization of millions of molecules • Proper communication between these molecules is essential to the normal functioning of the cell • To understand communication between molecules:*determine the arrangement of the atoms*

4. Advanced Cell & Developmental Biology

5. Gene, Recombinant DNA & Cloning Analysis

6. Restriction Enzymes • Restriction enzymes are DNases (nucleases) found in bacteria that recognize specific DNA sequences as 4mers,6mers or 8mers and make double stranded breaks in DNA . • This enables cutting of genome in specific ways to generate restriction site maps and the development of approaches for pasting pieces of DNA together in specific ways. A Separation of EcoR1 segments on an agarose gel B C D ,E F

7. DNA Hybridization • DNA hybridization is the process whereby complementary strand of DNA anneals (to form a double helix) with the single stranded DNA • Hybridization can be measured by labeling the “complementary strand” either with 32P nucleotides or fluorescent probes . • There is also DNA-RNA hybridization

8. Cut DNA with restriction enzymes Separate fragments on agarose or acrylamide gels Transfer the separated DNA from gel on to nitrocellulose paper Southern Blotting • Southern Blotting enables identification of specific DNA sequences (gene • fragments) from among the total sequence of DNA Hybridize with a labeled DNA or RNA of interest ( e.g., 32P labeled DNA) followed by autoradiography or phosphoimaging for detection

9. Northern Blotting • Northern Blotting is where RNA is blotted and then probed labeled DNA (cDNA) • synthesized from the mRNA isolated from the cell • Enables identification and quantification of specific mRNAs from among the vast • population of RNAs in the cell

10. DNA cloning • DNA cloning enables specific pieces of genome to be inserted into bacteria as plasmid or phage lambda vectors and grown in large quantity. • The first step is to generate a library of bacteria with inserted DNA fragments. This could either be a genomic(DNA)or a cDNA (mRNA) library

11. Replica plating and in situ hybridization • Techniques used to identify a bacterial colony that contains the gene (DNA sequence) • of interest. The isolated colony can be grown up in large quantities. CsCl centrifugation for separation of plamid DNA from chromosomal DNA Replica plating and in situ hybridization

12. cDNA libraries • They are generated to isolate particular genes of interest or to identify a gene based on the protein expression of that gene cloned in the bacterial cell • The latter procedure is called “reverse genetics” whereby the protein product is used to identify the gene followed by DNA sequencing

13. DNA sequencing • Sanger’s dideoxy method DNA to be sequenced is mixed with each of 4 ddNTPS (chain terminators) in separate reactions for DNA synthesis and later separation of the products by electrophoresis • Can now be done automatically via sequencing machines that work with different flurochromes attached to each of dideoxy nucleotides • To determine the sequence of a gene of many kilobases overlapping DNA fragments of 400-800 bp must be sequenced

14. Protein expression vectors • These are specially designed plasmid • vectors for fusion protein expression • to isolate large quantities of protein of • interest for antibody production or • other studies of purified protein. • The proteins are produced as fusion • proteins of the cDNA gene coding • sequence ligated to a protein • expression marker or reporter protein • e.g. beta-galactosidase • They can also be used as a major tool • in cell biology to study the expression • of proteins in cells following DNA • transfection

15. DNA transfection and Polymerase chain reaction (PCR) • DNA transfectionis used to track the properties of individual proteins in a cell • Construct a plasmid expression system that contains the protein of interest fused with a reporter gene such as a beta- galactosidase or a short peptide sequence such as HA 9 mer peptide or FLAG epitope for antibody localization with anti HA or anti FLAG or fluorescent localization in living cells with GFP-constructs (GFP-actin) Polymerase chain reaction (PCR) Is used as an alternative to cloning for purifying a particular DNA (gene sequence It enables the production of microgram quantities of the DNA sequence of interest in the test tube Provides an alternative for preparing DNA probes to screen genomic or cDNA library for clones encoding a protein of interest

16. DNA Microarrays and chips • Enable via fluorescence in situ hybridization (FISH) to measure expression of 1000’s of genes on each array/ chip. Actual chip size Yeast genome microarray: The array is hybridized to cDNA labeled with a green fluorescent dye prepared from cells grown in glucose and with red labeled cDNA from cells grown in ethanol. Spots were detected with a scanning confocal microscope

17. Antibody production • Polyclonal antibodies are • generated by injecting • antigen into an animal and • purifying the antibody • titer from blood • Monoclonal antibody • technique enables to obtain • a single clone of cells that • recognizes one epitope • ( usually ~ 9 a.a.) of the • total protein Monoclonal antibody production

18. Genetic Engineering • Introduction of exogenous genes ( mutant or normal) in to normal cells or organisms to study gene expression • Used to study the role of the protein coded by the gene in the cell/organism function or for engineering gene expression for improving food production or reducing the destrcutive damage of human diseases

19. Site Directed Mutagenesis • Alterations in nucleotides (substitutions or deletions) in vitro at known (directed) sites to create “mutant genes” • These mutant genes can be transfected into cells as previously discussed and enables study of gene function at the individual cell level. The transfected genes are also called “transgenes”

20. Production of transgenic mouse Inject mutant gene in to one of the pronuclei of the fertilized mouse oocyte Transfer oocyte to surrogate mother. 10-30% of offspring contain the transgene in equal amounts in all tissues

21. Gene Knockout or “replacement” • Form of trangenics • Occurs following homologous recombination of the transgene at the site of the endogenous gene • Occurs readily in yeast cells but in mammalian cells the rate of recombination is very slow and hence a double selection marker approach is adopted where the first marker e.g. neomycin resistance selects for all cells with homologous recombination while the second marker allows growth of only those cells that carried out homologous recombination

22. Knockout protocol ES cells are isolated from the inner blastocyst and culture ES cells are tranfected with the gene of interest Enables direct study of gene function in an intact organism ES cells successfully transfected via homologous recombination are selected and grown in culture and injected into a host blastocyst. Chimeras develop which contain ES cells from both the transfected and the host cells.

23. Gene Replacement/therapy • Replace an abnormal • gene with a normal one • at a very early stage of • development • It has the potential for • curing or alleviating the • symptoms of a wide • variety of human • diseases, e.g.,Parkinson’s • disease Procedure for gene replacement

24. Mammary gland cells Finn Dorset ewe 3.5 months pregnant Harvest quiescent cells How Ian Wilmut Made Dolly 1Making Quiescent Cells Culture mammary cells Starve cells

25. Glass pipette Suction Pipette Suction How Ian Wilmut Made Dolly 2Collecting The Donor Nucleus

26. Suction Pipette Suction How Ian Wilmut Made Dolly 2Collecting The Donor Nucleus Glass pipette

27. Egg Scottish Blackfaced ewe egg donor How Ian Wilmut Made Dolly 3Egg Preparation An egg is collected then placed into a dish where it can be manipulated

28. Glass pipette Chromosomes Suction Pipette Suction How Ian Wilmut Made Dolly 3Egg Preparation Egg

29. Chromosomes Suction Pipette Suction How Ian Wilmut Made Dolly 3Egg Preparation Glass pipette Egg

30. Glass pipette Suction Pipette Suction How Ian Wilmut Made Dolly 4Inserting The Donor Nucleus

31. Suction Pipette Suction How Ian Wilmut Made Dolly 4Inserting The Donor Nucleus Glass pipette

32. Suction Pipette Suction How Ian Wilmut Made Dolly 4Inserting The Donor Nucleus

33. How Ian Wilmut Made Dolly 5Initiating Development

34. How Ian Wilmut Made Dolly 5Initiating Development Zygote

35. How Ian Wilmut Made Dolly 5Initiating Development Cleavage

36. How Ian Wilmut Made Dolly 5Initiating Development Cleavage

37. How Ian Wilmut Made Dolly 5Initiating Development Cleavage

38. How Ian Wilmut Made Dolly 5Initiating Development Cleavage

39. How Ian Wilmut Made Dolly 5Initiating Development Morula

40. Morula Scottish Blackfaced ewe surrogate mother Finn Dorset lamb Dolly How Ian Wilmut Made Dolly 6Development