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DNA Transformations in C. elegans Using MosSCI Whitney Evans.

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Presentation on theme: "DNA Transformations in C. elegans Using MosSCI Whitney Evans."— Presentation transcript:

1 DNA Transformations in C. elegans Using MosSCI Whitney Evans

2 Outline 1.C. elegans Facts 2.Previous Research – Fire and Mello 3.Previous Research – Jorgensen 4.Proposed Plan and Materials and Methods

3 Caenorhabditis elegans 1974 – Sydney Brenner began using C. elegans as a model organism -Animal behavior and development

4 Caenorhabditis elegans Rapid Life Cycle Small size Ease of Laboratory cultivation

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6 Caenorhabditis elegans Grown in petri dishes with TSB media – NaCl3 g Peptone5 g Bacto-Agar17 g Deionized H2O1000 ml 5mg/ml Cholesterol in 95% ethanol1 ml 1M CaCl21 ml 1M MgSO42 ml 1M KPO425 ml On a lawn of Escherichia coli

7 Craig Mello Research ZKfqjhoM:&ved=0CAUQjRw&url=http%3A%2F%2Fthefutureofthings.com%2Fnews%2F1311%2Fworm-with- sight.html&ei=W0XpUZHyN4LlyAHZu4CoAw&bvm=bv ,d.aWc&psig=AFQjCNG8dfmP4YJSLYOC9pcCYTKhHxIGWw&u st=

8 Craig Mello Research Microinjection procedure for C. elegans DNA transformation Using a dominant-acting genetic marker – rol-6(su-1006) Heritability of transgenic extrachromosomal elements The exogenous DNA recombines with the worm’s DNA

9 Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10: 3959– Place the injection needle into the distal gonad 1.Begin pushing the injection mix into the distal gonad 1.As the injection mix fills the distal gonad, the mix will cause the gonad to swell

10 Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10: 3959–3970 # of PO F1# F1 w/rol F2

11 Richard R Zwaal, Julie Ahringer, Henri G.A.M van Luenen, Alice Rushforth, Phil Anderson, Ronald H.A Plasterk, G Proteins Are Required for Spatial Orientation of Early Cell Cleavages in C. elegans Embryos, Cell, Volume 86, Issue 4, 23 August 1996, Pages , ISSN , 8674(00)80135-X. (http://www.sciencedirect.com/science/articl e/pii/S X)

12 Jorgensen Lab Research MosSCI: mos1 single copy insertion

13 Frøkjær-Jensen, C., Davis, M., Hopkins, C., Newman, B., Thummel, J., Olesen, S., Grunnet, M., & Jorgensen, E. (2008). Single copy insertion of transgenes in c. elegans. Nature Genetics, 40(11), doi: /ng.248

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15 Mello Injected mutant gene rol- 6(su-1006) Into wildtype worm (N2) Multiple insertion sites Jorgensen Injected wildtype gene unc- 119(+), along with a repair template, transposase, and fluorescent positive and negative selection markers Into mutant worm (EG6699 or EG4322) Single insertion site

16 Which has a better rate of transfection: Multi-site insertion (Mello) or Single-site insertion (Jorgensen)?

17 Proposed Plan 1.Acquire capability in injecting 2.Collect data on transfection rate of multi-site insertion 3.Create plasmid for single-site insertion using the Gateway Method 4.Collect data on transfection rate of single-site insertion 5.Compare multi-site and single site transfection rates

18 Isolating DNA Perform Plasmid Prep Check Concentration using Nano Drop

19 Nano Drop

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22 Microinjection pRF4 Three worms are place vertically in mineral oil on an agarose cover slip. One drop of mineral oil is used. Each worm is injected at both distal gonads. After the worms are injected with the plasmid a drop of M9 buffer is placed on the cover sheet and engulfs the worms. The coversheet is then place in a water chamber for 1 hour. Then, each worm is transferred to individual Petri dishes seeded with OP50. pRF4 Three worms are place vertically in mineral oil on an agarose cover slip. One drop of mineral oil is used. Each worm is injected at both distal gonads. After the worms are injected with the plasmid a drop of M9 buffer is placed on the cover sheet and engulfs the worms. The coversheet is then place in a water chamber for 1 hour. Then, each worm is transferred to individual Petri dishes seeded with OP50. pRF4 Three worms are place vertically in mineral oil on an agarose cover slip. One drop of mineral oil is used. Each worm is injected at both distal gonads. After the worms are injected with the plasmid a drop of M9 buffer is placed on the cover sheet and engulfs the worms. The coversheet is then place in a water chamber for 1 hour. Then, each worm is transferred to individual Petri dishes seeded with OP50. pRF4

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24 Date Concentration (ng/μl) # Worms Injected # Survived Injection% Survived# with Rol F1 % Produce Rolling F1 2/21/ ND 00 3/10/ /14/ /15/ /16/ /16/ /20/ /21/ ND0 4/22/ ND0 4/23/ ND0 5/11/ /17/ /19/ /1/ /17/ /20/ /22/ /2/ Total WormS

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29 Heritability

30 Mutant Rescue Using pLB10

31 Unc Rescue Total Injected# Survived# Rescued# Partial Rescue# Non Rescue% Full Rescue% Full + Partial Rescue

32 Mos1 is a transposon that can be easily located in the genome. The sight that is primarily used is on Chromosome II at the ttTi5605 locus. Gateway Technology can be used to construct the extrachromosome array, which includes positive and negative selection markers, the gene of interest, and the transposase. The Mos1 is excised by the transposase, resulting in a double-strand break in the chromosome. The 3’ ends of the left (L) and right (R) flanks anneal to the homologous regions of the extrachromosomal array. The break is repaired by synthesis-dependant strand annealing. The positive selection marker and gene of interest are now inserted into the genome. unc-119 (+) is often used as a positive selection marker. This requires an unc-119 background and successful injections are determined by unc-119 rescue. Mos1 is a transposon that can be easily located in the genome. The sight that is primarily used is on Chromosome II at the ttTi5605 locus. Gateway Technology can be used to construct the extrachromosome array, which includes positive and negative selection markers, the gene of interest, and the transposase. The Mos1 is excised by the transposase, resulting in a double-strand break in the chromosome. The 3’ ends of the left (L) and right (R) flanks anneal to the homologous regions of the extrachromosomal array. The break is repaired by synthesis-dependant strand annealing. The positive selection marker and gene of interest are now inserted into the genome. unc-119 (+) is often used as a positive selection marker. This requires an unc-119 background and successful injections are determined by unc-119 rescue. Mos1 Transposon

33 Transposase

34 Homologous Recombination

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37 pLB10

38 Mos transposase

39 Remaining Aspects of Project Use Gateway Method to Create Plasmid Compare Multi-site insertion to Single-site insertion

40 Evans, T. C., ed. Transformation and microinjection (April 6, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/ /wormbook , Frøkjær-Jensen, C., Davis, M., Hopkins, C., Newman, B., Thummel, J., Olesen, S., Grunnet, M., & Jorgensen, E. (2008). Single copy insertion of transgenes in c. elegans. Nature Genetics, 40(11), doi: /ng.248 Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10: 3959–3970 Padmanabhan V, Khan ZS, Solomon DE, Armstrong A, Rumbaugh KP, et al. (2012) Locomotion of C. elegans: A Piecewise-Harmonic Curvature Representation of Nematode Behavior. PLoS ONE 7(7): e doi: /journal.pone Richard R Zwaal, Julie Ahringer, Henri G.A.M van Luenen, Alice Rushforth, Phil Anderson, Ronald H.A Plasterk, G Proteins Are Required for Spatial Orientation of Early Cell Cleavages in C. elegans Embryos, Cell, Volume 86, Issue 4, 23 August 1996, Pages , ISSN , X.

41 Questions


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