Transcript notes
Berthold’s Experiment: Introduction • Background: pangenesis/preformation theory of reproduction – Idea that various body parts secreted bits of themselves into the blood and these assembled in semen. Preformation extends this to teeny animals (homunculi) being formed in sperm in the testes. These homunculi then simply grew larger to form babies. • Not a very promising start, but it had some truth that helped Berthold: – Body parts able to secrete factors into blood – These factors have targets distant to origin – The testes were somehow involved in this Berthold’s Hypothesis • According to pangenesis/preformation, bits of organs travel through the blood stream to the testes to make a copy of that animal. This theory makes a specific prediction that seems testable: Hypothesis: transplanted testes should pass on the properties of host and not the donor to offspring… (N.B. this is my guess as to why Berthold did his experiment, we don’t really know why) Berthold’s Experiment: Methods • Experimental design: – control group 1: castrated only Concern: What are effects of castration on roosters? – control group 2: castrated and testes reimplanted Concern: Will testes be functional after removal and implantation? – experimental group: castrated and cross- implanted Will testes function like the host (support pangenesis) or like the donor (refute pangenesis). Unfortunately for our imagined hypothesis, implanted roosters aren’t fertile. So we get no further towards disproving pangenesis. BUT… serendipity! TARGET PROBE LABEL IN SITU IN VITRO Hormone (nonprotein) Antibody Isotope Enzyme Protein Antibody RNA cDNA Isotope ISH Enzyme Fluorophore Northern RT-PCR microarray DNA cDNA Isotope Not common Enzyme Fluorophore Southern PCR RFLP Not RIA Common EIA/ELISA Enzyme Assays Isotope IHC Western Enzyme (not Fluorophore ICC!) Microarrays • Relatively new technology. • Different kinds, idea is that the expression of 1000s of genes can be determined at once using an array of very small dots, each of a specific cDNA • This kind of “high throughput” sampling of gene expression is very fashionable Benefit: lots of information fast Cost: expensive, validation and analysis is laborious, often inconclusive (fishing expedition) Genetically modified organisms • Transgenesis (designer genes) Artificial genes that are added to genome • Gene Targeting (knock-outs/ knockins) Artificial genes that replace genomic sequences Benefit: very powerful way of testing gene function Cost: laborious & time intensive, abnormal genetic makeup often complicates study Temporary genetic manipulations • Antibody binding can be used to interfere with protein function as well as for detection • RNAi = RNA interference (a.k.a. antisense). cRNA or cDNA can reduce protein abundance of specific genes • Transfection (a.k.a. gene therapy). Introduction of foreign genes into cells using vectors (eg viruses) Benefits: less time-intensive than GMOs, can also overcome developmental and compensatory effects often seen in GMOs Costs: transient, variable, inefficient