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

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

3.  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:  To understand communication between molecules: *determine the arrangement of the atoms* Organ  Tissue  Cell  Molecule  Atoms Structural Biology Medicine and Biology at the Atomic Scale

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 B C D,E F Separation of EcoR1 segments on an agarose gel

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 32 P nucleotides or fluorescent probes. There is also DNA-RNA hybridization

8. Southern Blotting Southern Blotting enables identification of specific DNA sequences (gene fragments) from among the total sequence of DNA Cut DNA with restriction enzymes Separate fragments on agarose or acrylamide gels Transfer the separated DNA from gel on to nitrocellulose paper Hybridize with a labeled DNA or RNA of interest ( e.g., 32 P 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. Replica plating and in situ hybridization CsCl centrifugation for separation of plamid DNA from chromosomal DNA

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 transfection is 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. 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 Actual chip size

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 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. Enables direct study of gene function in an intact organism

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

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

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

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

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

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

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

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

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

33. How Ian Wilmut Made Dolly 5 Initiating Development

34. Zygote How Ian Wilmut Made Dolly 5 Initiating Development

35. Cleavage How Ian Wilmut Made Dolly 5 Initiating Development

36. Cleavage How Ian Wilmut Made Dolly 5 Initiating Development

37. Cleavage How Ian Wilmut Made Dolly 5 Initiating Development

38. Cleavage How Ian Wilmut Made Dolly 5 Initiating Development

39. Morula How Ian Wilmut Made Dolly 5 Initiating Development

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