Ifferentiation in Caenorhabditis elegans i??i??
(During animal development cell division generates large numbers of cells and subsequently these cells differentiate to follow specific fates. E.g. a nerve cell is born as an undifferentiated cell and only demonstrates the particular characteristics, by which we identify the cell as a nerve cell, later. Differentiation depends on expression of the set of genes which encode the proteins which will confer all the appropriate activities to the mature cell. E.g. a nerve cell must produce enzymes for synthesis of neurotransmitters. Differentiation can be observed using a reporter gene to report on the regulation of one member of the set of genes to be expressed in a particular cell type.
i. One commonly used reporter gene is lacZ, a bacterial gene encoding the enzyme AYgalactosidase. This enzyme is readily expressed in animal cells and sites of expression can be determined easily by histochemical staining. X-gal, a colourless substrate of AYgalactosidase, is cleaved to give an insoluble blue product which precipitates at the sites of AY-galactosidase activity.
You will be provided with two strains, UL1 and UL6, of the nematode worm Caenorhabditis elegans. You will work with one of these strains and your partner will work with the other. The lacZ gene is present in both strains but has been fused to the regulatory elements of different genes so AY-galactosidase will be expressed in different cells in the two strains. You will stain these two strains for AY-galactosidase, describe the patterns of AY-galactosidase expression and interpret what you are observing.
1. Wash the worms from each plate into a separate eppendorf tube using about 1ml of distilled water and a Gilson pipette.
2. After 5 minutes the worms should have settled to the bottom of the tube and 3I?l aliquots containing the worms may be removed to each well of a multiwell glass microscope slide. Use one slide for each worm strain, labelling the slides with a pencil.
3. Place a coverslip on each slide over the worm aliquots and place the slides on the metal plate on dry ice. This snap freezes the worms and allows penetration of the stain.
4. After 3 minutes, flick off the coverslips with a razor blade and fix the specimens by immediately placing the slides in acetone at -20oC. The slides should not be allowed to thaw before placing in the acetone, so place rapidly in the acetone and do not put the slides on the bench as the transfer of heat will be fast enough to thaw the slides.
5. After 5 minutes, remove the slides and allow to air dry on the bench.
6. Place 3I?l of X-gal staining solution in each well of the slide. Lower a coverslip onto the
slide using the razor blade, smoothly, avoiding air bubbles. Holding the cover slip down to stop it moving, seal with nail varnish around the edges of the cover slip. Do not lift the coverslip after you start to lower it, or the worms will be lost from the slide.
7. Incubate 1hr at 37oC. (During this incubation you should look at the GFP expressing worms as described on the next page and look at the plates you set up last week, recording your observations/results.)
8. Observe your stained specimens carefully and very closely under the microscope. 9. Interpret your observations in terms of development. This needs careful, detailed
observation and thought, relating your observations to development in terms of spatial changes over time from fertilization through embryogenesis and larval stages to the adult form. You do not have enough time to see all that there is to see in the slides you have generated but take as much time as you have available to study your slides and think carefully about how what you are seeing relates to animal development. Micrographs and cartoons on page viii explain a little of the development and anatomy of this species to help you interpret your observations.
10. Draw diagrams of what you are observing to illustrate your interpretations.)
writing styles instruction :
Generic Feedback on Full Practical Report
Substantial individual comments have been placed on the marked reports. The generic comments, provided below, identify common issues and may well reinforce some of those individual comments. Professor Isaac also identified many of these points in the generic feedback he provided on the reports that he marked.
Despite being directed to BioScience Horizons most students failed to follow the a?Instructions to Authorsa?. If you have not looked at some of the articles published in this journal, all written by undergraduates like yourselves, I strongly encourage you to do so. Please observe the writing style and format. All scientific journals use formats very similar to that in BioScience Horizons and you should be reading such articles each week such that thinking/writing this way is second nature.
Many reports did not include an Abstract, despite me pointing out that this was the major failing of the example provided to you as good practice, through the VLE.
The background included within the Introduction should be relevant to your results and/or conclusion. It is not appropriate to simply write all that you know on the subject. The introduction sets the context for your results.
The Methods section is an account of precisely what you actually did. A common mistake still is to write the Methods section as instructions for somebody else to follow. This reflects a misunderstanding of the requirement to a?write the Methods section such that somebody else could repeat the worka?. This means that you need to describe what you did in sufficient detail that somebody else could make the same observations independently. Look at scientific papers and see for yourself.
Results sections in scientific papers are typically the longest sections of text. The text description of your observations refers to the figures or tables to clarify/illustrate/provide more details of what you have written in the text. A results section does NOT consist simply of figures or tables. All figures and tables should be referred to from the main text by number. Figures and tables are typically referred to from within brackets at the end of a sentence, not as the subject of a sentence. E.g. a?The embryo was small (Fig.1)a?, and NOT a?Figure 1 shows the embryo was small.a? Each figure should have a number, title and legend, and source acknowledgement if derived from somebody else (e.g. ..(Adapted from Smith and Jones, 2010.). Each table should have a number and title. A detailed results section depends on careful, in-depth observation of the results in the laboratory and many of you did not look closely enough at the biological material you were provided with or generated yourself.
The Discussion should relate your observations, as described in the Results section, back to the background you provided in the Introduction. This may include alternative interpretations of your data which are not strongly supported by your results.
Referencing did not follow the journal style, as directed in the Instructions to Authors. Some reports cited no references at all. While citations are found extensively in scientific writing, quotations are rarely used. Quotations only appear if the particular turn of phrase is important.)