LATG Chapters 8 & 9

1. LATGChapters 8 & 9 Molecular Biology and Genetics

2. Molecular Biology • …is the study of biology at the molecular level • …focuses specifically on DNA, RNA, and protein • …is a tool used to study genetics

3. SomeDefinitions • Genetics …is the study of how genes interact (with each other AND their environment) to produce the inherited characteristics that we see every day • Genome…the entire collection of genes an organism has.

4. More Definitions: • Genotype = the genetic makeup of an organism • Every person (every mouse, every cow) has two copies of each gene, one from each parent • “Homozygous normal” = two normal copies (aka Wildtype) • “Heterozygote” = one normal & one abnormal copy • “Homozygous abnormal” = two abnormal copies (in transgenics, aka “Knock-Out”

5. There’sstill more... • Phenotype= the physical features of an organism (i.e., tall/short; red/white etc) • Mutation= any change in the DNA of a gene • Genetic Engineering...is the term used to describe the manipulation of the genetic make-up of an organism

6. Where is your DNA located?

8. Where is your DNA? • DNA is in the nucleus of the cell, on structures called chromosomes. • Chromosomes are made of genes • Genes are made of DNA

9. Structure of DNA • DNA is a long string (polymer) of 4 bases • These bases universal! • A = Adenosine • T = Thymine • C = Cytosine • G = Guanine • The order (sequence) of the bases is what makes one gene different from another gene.

10. DNA Structure, cont’d • A -- T • C -- G • GATTCC • CTAAGG • DNA exists as a double helix (twisted ladder) • Each rung of the ladder is a base pair

11. How do cells transmit their genetic information? • Replication = the process by which a cell duplicates its DNA. • When a cell divides,one copy gets passed onto the new cell

12. How do cells interpret the information in the DNA? • Transcription: the process by which the DNA code is “read”. • DNA is transcribed into RNA in the nucleus

13. Transcription • RNA has 4 bases: • Adenine A=U • Guanine G=C • Cytosine • Uracil*** (Uracil is used instead of Thymine) • Unlike DNA, RNA is single-stranded

14. Translation • Occurs outside the nucleus, via ribosomes • RNA is “read” in groups of three bases called “codons.” • Each codon corresponds to an amino acid

15. Information Flow in the Cell

16. Techniques • Extraction of DNA • Restriction Digestion • PCR • Electrophoresis • Southern blotting

17. Extraction of DNA • Enzymes “digest” cell walls and release DNA into solution • Add phenol to remove proteins • Spin to separate DNA from proteins • Add ethanol

18. Restriction enzymes • These are enzymes that cut (digest) DNA at specific sites (sequences). • Examples: • Eco RI only cuts the sequence …GAATTC… • Pst 1 only cuts the sequence …CTGCAG • Because everyone’s DNA is comprised of the same 4 nucleotides (A,T, C, G), you can attach one species to another...

19. Cloning (using RE)

20. Polymerase Chain Reaction (PCR) • Based on the fact that A=T and C=G • Need only a tiny bit of DNA • ***Must know some of the sequence of the gene of interest • Three simple steps: heat, add primers etc, cool solution so bases will bind, repeat! • Can amplify a piece of DNA a million-fold!

21. PCR

22. Detection of DNA • Use agarose gels • Gel acts like a filter: DNA separates by size • Stain the gel with a dye to make the DNA to make it fluoresce under UV light.

25. Blotting • Southern Blot • DNA cut with enzymes • transferred to membrane • hybridized to probe that is specific to gene of interest • exposed to film • if gene is present, you will see a band on the film

27. Types of Blots • Southern blotting -- DNA • Northern blotting -- RNA • Western blotting -- protein

29. Genetic Engineering of Mice • Two types • Transgenic - a gene is added via pronuclear injection • This is used to “overexpress” a gene • ex: Alzheimer’s and Beta-amyloid • Targeted Mutation (aka “KO”) - a gene altered then added to the genome using ES cells • This is used to delete a gene • ex: ERKO--estrogen receptor KO mice

30. Transgenic mice • Created through pronuclear injection • Need 4 groups of mice • superovulated females • stud males • vasectomized males • pseudopregnant females

31. The Mice • Superovulated females --given hormone injections to make them release more eggs than usual (30-60) • Stud Males --are mated with the s.o.females so that a lot of embryos are produced • Pseudopregnant females --a female is mated to a sterile male so that her body will produce hormones that prepare it for pregnancy

32. Procedure

34. Transgenic Mice • Transgene can integrate ANYWHERE in the mouse genome. • Integrates in 1-several hundred copies • Must screen pups (PCR) to determine which pup have the transgene, and will pass it on. • Must observe transgenic mice carefully to observe phenotype

35. Phenotype • Depends on the gene you’re overexpressing • Sometimes it’s obvious, sometimes it’s not. • Must observe these mice carefully

36. Knockout Mice • Knockout mice --a gene is deleted • Similar to transgenic mice, must carefully observe for phenotype • Need the same 4 groups of mice, plus ES cells • ES cells = embryonic stem cells • totipotent

37. Embryonic Stem Cells • Are from the inner cell mass of a blastocyst • can develop into any part of the body • “totipotent”

40. KnockOut Mice • Culture ES cells from white (129) mice and target gene using electric current • Mate black mice; insert “white” ES cells into black blastocyst • Pups = chimeras (black and white) • Mate chimera to black mouse. • If white pups are produced, targeted gene has been passed on!

41. Transgenic Animals and You • You are very important!! • These mice are expensive/time-consuming to make • You must carefully observe the animals for phenotype changes • strange gait, spinning, too fat, too thin, scaly skin…

42. Good Luck!