Cloning

1. Cloning • A clone is an exact genetic copy of an individual • Many organisms have clonal reproduction: fission (bacteria, protozoa), budding (some plants, invertebrates), parthenogenesis (some fish, insects, lizards) • Clonal reproduction of plants, using cuttings or other culturing techniques, mastered by humans for millennia (e.g. bananas)

2. Asexual Organisms A few species, like this gecko, are parthenogenic. This means that all individuals are female, and their offspring are exact genetic copies of themselves, or clones.

3. Asexual Organisms Most asexual species have close relatives that are sexual. The whiptail lizard is a parthenogenic species that formed as a hybrid of two other species. It tricks males of the other species to mate with it, because it needs sperm to activate its eggs

4. Cloning • Vertebrate cloning difficult – especially mammals & birds • Dolly was 1of 276 attempts • Current technology: • Harvest the nucleus of a cell from an adult • Inject it into an egg cell that has had its nucleus removed • Make sure that cells begin to divide normally (an embryo begins to form) – This is the hard step • Place the developing embryo in a foster mother

6. Cloning • Cloning (potentially) permits the rapid, economical reproduction of individuals with desirable traits • With serial cloning, genetic lines can be preserved (almost) exactly • Serial cloning may destabilize chromosomes • Mutations will accumulate – a cost of clonal reproduction • Even though the nucleus of the egg is removed, some of the egg-donors genes remain, in the mitochondria – thus the first generation is not a perfect clone • Long term risk in terms of lost genetic diversity • More vulnerable to disease epidemics • Less variation to adapt to environmental change

7. Gene ‘Therapies’ • Introduce substances that halt the activity of certain genes (AZT) • Introduce substances that (over) promote activity of certain genes (gamma globin) • Introduce new, functional genes true Gene Therapy • Experimental, currently tested primarily on critically ill patients

8. Marker Gene Therapy • Vehicle or Vector • e.g. weakened virus • Recipient cells • cultured cells or in vivo somatic cells e.g bone marrow • The introduced sequence • Marker (so you know it worked) • Promoter (to cause transcription) • The Gene

9. The Problems • Must immuno-suppress the patient • Efficiency of transfer often low • Duration of expression often short • Unknown risks… permanent unwanted changes (mutations) to somatic (or germ cells), viral infections, cancer or other diseases associated with breakdown in genetic regulation?

10. Genetically Modified (Transgenic) Crops • 50% of soybeans, 25% of corn grown in the US are transgenic (have a gene from another species added via biotech) • Main transgenic traits: herbicide tolerance, Bt toxins to kill insect pests, virus resistance • Future transgenic traits: vitamins, vaccines (inactivated viruses) • Potential to reduce uses of agrochemicals, increase food production, improve quality of plant products (including novel ones)

11. Genetically Modified (Transgenic) Crops • Using specialized enzymes, chop up gene sequences, select and connect desirable sequences to form a ‘cassette’ • Sequence must include a marker, promoter, transgene • Flanked by ‘sticky ends’ • Infect a bacteria (E. coli) to produce quantities of cassettes via bacterial clonal reproduction • Purify sequences, inject sequences into cells • Gene gun • Infective bacteria • Select plant cells with the marker • Propagate selected plants clonally • Produce new plants clonally or through sexual crosses

12. Marker

13. Transgenic Non-transgenic

14. Problems with Transgenic Crops • Doesn’t work very well - yet • Risk of traits spreading to weeds, producing ‘super weeds’, or pests developing resistance to transgenic defenses • Risk to non-target organisms • No unusual health risks (?), requires strict testing • Reduced genetic diversity puts crops at (long term) risk • Agricultural resources become monopolized by a small number of gigantic agrobusinesses

15. Transgenic Animals Potentially quite useful • Animal models of human diseases by introducing human genes • Targeted production of pharmaceutical proteins (human enzymes, hormones, growth factors) • Modification of animal anatomy & physiology

16. Transgenic Animals • Create ‘cassette’: enhancer + promoter + gene plus sticky ends • Enhancer often designed to restrict activation of the gene to a targeted tissue (e.g liver) only • Infect a bacteria (E. coli) to amplify the cassettes via bacterial clonal reproduction • Collect and purify the cassettes, test for activation in eukaryotic cells • Inject cassettes into a newly fertilized egg cells • Transfer embryos into a surrogate mother • Analyze babies, breed or reproduce clones of those that express the transgene and have the fewest other problems

18. Problems with Transgenic Animals • Doesn’t work very well – yet • Cassettes integrate randomly into genome, sometimes knocking out genes • Sometimes multiple copies integrate • Integration not always stable • Same worries as transgenic crops • Animal welfare