Identification the Stages of C elegans - Lab Report Example

Identification the Stages of C elegans ABSTRACT C.elegans is an important model organism. Gene expressions maybe studied on this model organism. The aim of this experiment is to identify the stages of C.elegans and understand the role of RNAi in producing mutant C.elegans. For this experiment we picked mutant types with the genes dpy-10,rol-5,bli-1 and unc-22 silenced. This silencing of genes with the process of RNAi helps to produce mutants. The wild type and the mutants are observed. The mutants explain the role of the genes and the protein expression in the wild type and hence are essential for genetic studies. The latter part of the experiment ensures learning about the techniques to culture C.elegans in a laboratory set-up. Introduction Most molecular genetics experiment cannot be employed on humans because of their complexicity. In order to perform these experiments researchers use certain model organisms which can be cultured very easily in the laboratory and is easy to manipulate. These model organisms though physically different from humans bear certain biochemical and physiological features that have been conserved during evolution (Hedges,2002, p838). With this respect the nematode worm Caenorhabditis elegans offer certain exceptional advantages and is one of the foremost model systems in molecular genetics. In order to study gene function in model organisms single gene mutations are introduced and phonotypical characters are analyzed. The result of this analysis may vary-some of these single gene mutations would produce drastic biochemical or structural differences while others may go silent when compared with the wild type phenotype (Fire at al,1998).. Background of RNAi RNAi (RNA inference) also known as post-transcriptional gene silencing is a biological process which down regulates expression of the targeted gene . Exogenous dsRNA could induce potent and sequence specific silencing of endogenous gene expression in C.elegans (Fire et al,1998) i.e. intentional introduction of dsRNA into any organism, the difference in traits as a result of gene silencing maybe observed. Worms as a model system: Most molecular genetics experiment cannot be employed on humans because of their complexicity. In order to perform these experiments researchers use certain model organisms which can be cultured very easily in the laboratory and is easy to manipulate and C-elegans is an ideal organism because of the following characteristics: It is an eukaryote Genome size is small (97 Megabases) Small lifecycle Easy to maintain in laboratory (Gilbert,2000) Gene that we studied: The entire genome of C.elegans has been sequenced and therefore we have a clear understanding about the genes. We used 4 mutant with different genes suppressed so as to try and understand the role of the genes in the wild C.elegans. The genes are dpy-10, rol-6, bli-1 and unc-22. OBSERVING WILD TYPE AND MUTANT C.ELEGANS AND CULTURING Laboratory 1 Material Required: Mutant worms on NGM-lite plates Wild-type worms on NGM-lite plates Binocular dissecting microscope Methods: 1. LAB1-part 1- Plates with wild worms are seen under microscope and behavior is observed. Life stage of microscope and drop on plate to induce movement. Tapping the plate may also be required to induce movement. Observation of lifecycle is made. 2. LAB1-part 2 Both mutant and wild types are observed under microscope and morphological and locomotor differences between them are observed and recorded. LABROATORY 2 E. coli are used as the food source for C. elegans. C. elegans are propagated on lawn of E coli on NGM-lite medium. The worms are propagated from plate to plate by the process of chucking and picking. Chucking and Picking Chucking is a rapid way of transfer and involved cutting out a portion of NGM-lite medium where E. coli food source had been consumed and transferring it on a new plate. Picking involves transferring of individual worms with the help of flattened tip of a platinum wire. Picking individual worms from particular stages is the first step of genetic crosses and RNAi knockdown. STEP 1-Chunking MATERIAL REQUIRED: OP50-seeded NGM lite plate Wild worms on NGM –lite plate Binocular dissecting microscope Bunsen burner 95% ethanol Scalpel/spatula/forceps METHOD: 1. The OP50 plate must be observed for contamination and only uncontaminated plate must be sued. 2. The plate containing wild worms is observed and the region where worms and eggs are seen most is identified. 3. Sterilized spatula or forceps is dipped in ethanol and ignited to kill microbes. Use this to cut the agar at the site where most worms and eggs were visible in the wild worm plate. 4. The cut agar is lifted and placed upside down on the fresh OP50 plate to enable the worms to crawl to their new food source 5. The OP50 plate is observed under microscope to ensure successful chucking 6. Plates are incubated at 20 degrees and stored for Step 2. Step 2-Picking Material required OP50-seeded NGM-lite plate Plate from step 1 (plate with wild worms) Microscope Bunsen burner Forceps Worm pick Bacteria plate METHOD: 1. The plate that was chunked in step 1 is examined for contamination. 2. A worm pick is taken and the platinum wire tip is adjusted (if need) at 45 degrees. 3. The tip of the pick is sterilized by holding it near the blue region of the Bunsen burner flame till it turns red hot. 4. Dip the wire in the bacteria plate to ensure a glob of sticky bacteria gets attached to it. These bacteria will act as a tape for worm picking. 5. The microscope is adjusted and focused on the wild worm plate such that the stages of the worm can be identified. 6. The plate lid is opened and a large adult warm is identified. 7. With the help of the worm pick the worm is gently picked and transferred into a fresh NGM-lite plate taking care not to crush the worm during the transfer process. 8. Observe under microscope to see if the worm has survived. This is repeated for practice purpose. 9. Once the hand becomes trained L4 worms are identified and transferred in the same manner into a fresh OP50-seeded NGM-lite plate. 10. Plate is incubated and stored. RESULTS 1. The lifecycle of C.elegans may be divided into 6 different stages namely embryo, larva1 (L1), larva2 (L2), larva 3(L3), larva 4(L4) and adult. During each larval stage the worm molts and continues to grow in size. We were able to 5 stages-embryo,L1, L2/L3,L4 and adult. L2 andL3 could not be distinguished positively. 2. It is necessary for C.elegans to pass through distinct phases before attaining sexual maturity since there is a physical limitation to growth during each stage. Human grow continuously and not in phases unlike C.elegans. Humans also grow through addition of cells and not molting. 3. A hermaphrodite has the sexual machinery of both genders. Therefore it can produce both sperms and oocyte. Sperms are produced in L4 stage approx 150 sperms per gonadal arm and then only oocyte is produced. Both sperms and oocyte are stored in the same gonadal region until the first oocyte pushes the sperm into the spermatheca where the oocytes are fertilized (WormAtlas, 2006). This is called self insemination. 4. Under the microscope while observing the mutant worms I observed a short and fat body. The wild type was active and moved on the agar surface but the mutant I observed rarely moved. No, my classmates did not identify the same characteristics. 5. I observed a short and fat worm body. Therefore I think that the protein that the wild type expressed must have been. Plasmid: To generate mutant C.elegans we need to introduce RNAi system in the cell therefore we need a vector for this transmission. In this case, plasmids are as vehicles to transport the RNAi system into the target cell. Mutant produced We used mutants dpy 10, rol-6, bli-1 and unc-22. Dpy 10- stand for Dumpy and gives rise to short worms. Rol-6- gives rise to mutant worms which appear circular. Bli-2 produces mutants which show a blister on one side Unc-22 mutants show twitching and their body is outstretched. CONCLUSION Phonotypical and behavioral changes are seen when protein fail to express thereby establishing the function of the protein. The genes that were silenced in the C.elegans mutants produced mutants which were different from the wild type C.elegans and also had different behavioral characteristics as well. References Fire, A, et al. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans." nature 391.6669 (1998): 806-811. Print. Gilbert, S F. Developmental Biology (6th Edition). Sinauer Associates Inc, 2000. Print. Hedges, S B. "THE ORIGIN AND EVOLUTION OF MODEL ORGANISM." Nature3.3 (2002): 838-846. Print. WormAtlas. "Hermaphrodite Introduction." Wormatlas Homepage. N.p., n.d. Web. 19 Nov. 2013. . Read More