JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

 JoVE Biology

Generation of Stable Transgenic C. elegans Using Microinjection

1, 1, 1, 1

1Department of Biological Sciences, University of Alabama

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 184.73.85.45, User IP: 184.73.85.45, User IP Hex: 3091813677

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

    Date Published: 8/15/2008, Issue 18; doi: 10.3791/833

    Cite this Article

    Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. Generation of Stable Transgenic C. elegans Using Microinjection. J. Vis. Exp. (18), e833, doi:10.3791/833 (2008).

    Abstract

    Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

    Protocol

    Expression Plasmid Construction

    Two plasmids are required: one for tissue-specific expression of the gene of interest and a second as selectable transformation marker.

    Experimental Plasmid

    1. Select a promoter that is expressed in the tissue/cell type of interest, for example, a nerve or muscle-specific promoter. If one is to express multiple genes within the same tissue, a promoter cassette plasmid in the Gateway system (Invitrogen) can be used. This approach allows the insertion of any gene of interest downstream of the promoter by recombinational cloning 4. Generally, 2-5 kb of upstream sequences is sufficient to have correct and accurate expression.

    2. The cDNA of the gene of interest can be cloned in two ways:
      1. Conventional cloning using restriction enzymes.
      2. For Gateway cloning, it is PCR amplified using primers that contain the Gateway att B recombinational sequence. The amplified cDNA is first recombined into the donor vector pDONR201 to create the entry vector and then transformed into E. coli strain DH5a. Following selection of successful recombinants and mini-prep isolation of DNA, the entry vector is recombined into the promoter-containing destination vector to create the expression plasmid. Details on recombinational cloning are available from either the Invitrogen Gateway manual or in Caldwell et al. 5.

    Selectable Marker Plasmid

    Examples of transgenic marker plasmids include rol-6 or use of the body wall muscle promoter unc-54 to drive fluorescent protein expression. These marker plasmids are usually available from within the research community upon request.

    Microinjection of C. elegans

    Preparation

    1. Perform DNA isolation of the two plasmids that are to be injected. Quantitate them and mix together in a 1:1 ratio of 50 ng/µl each.

    2. Prepare agar pads:
      1. Make a 2% agarose solution in water; heat or microwave until dissolved.
      2. Set out several 22 x 50 mm cover glasses on a benchtop.
      3. Using a Pasteur pipette, place a drop of the melted agarose on one cover glass and immediately place a second cover glass over the drop at a 90 angle to the first. Repeat for several other cover glasses. The diameter of the flattened disc of agarose should be between 15-20 mm.
      4. After the agarose has solidified (this only takes moments), remove the cover glass and allow the pad to air dry completely (several hours to overnight).
      5. Store the pads in a cover glass box at room temperature.

    3. Make needle loaders for DNA solution:

      Needle loaders are created from 100 µl capillary tubes that are heated in the middle over an open flame and rapidly pulled to approximately twice their length. The two halves are snapped apart once the capillary tube cools. Stockpile 10-20 needle loaders for injection. Store upright in a microcentrifuge tube rack. Use caution as to not impale oneself on the points.

    4. Make microinjection needles:

      The needle is made from a special capillary tube that contains an internal glass filament; this filament serves as a wick for the DNA solution. These are pulled on a needle-puller machine. We use a Narishige PP-830 puller with a heat setting of 24.8. Several needles are pulled at a time and are stored in a small plastic box. Multiple needles can be readily "mounted" on a strip of modeling clay for long-term storage.

    5. Needle tip "breakers":

      The tip of the microinjection needle is pulled so fine that it is actually closed at it end and should be broken open using small shards of cover glass. These are prepared by wrapping an 18 x 18 mm cover glass in a paper towel and gently applying pressure until it breaks into several pieces. Shards of a useful size are approximately 3 x 4 mm.

    6. Grow wild type (N2) worms to the early adult stage for injection using standard procedures 6. Prepare about 100 properly staged worms/construct injected.

    Procedure

    1. Open the main valve on a helium tank that is connected to a microinjection needle arm and holder. Close the regulator valve to allow the gas to enter the line, bringing the pressure to 35 psi. Notice that the gas can be released using a foot pedal.

    2. Insert a needle loader into a standard mouth pipetter tubing (found in containers of glass capillary tubes) and draw up a small amount of the plasmid mixture (~1 µl).

    3. The needle loader is carefully placed into the back end of an injection needle and gently inserted all the way through the length of the needle until resistance is felt. Expel the DNA solution by gently blowing, then remove the loader.

    4. Insert the needle into the microinjection arm, being careful to place it within the small internal rubber gasket.

    5. Place a needle-breaking shard of cover glass on one edge of an agar pad. Cover the shard, as well as the whole surface of the agar pad, with halocarbon oil. Place the agar pad on the stage of an inverted microscope.

    6. Position the needle in the center of the viewing field using the 4X objective and bright field illumination, but do not yet lower it into the oil. Also, position the inner edge of the shard of cover glass in the center, then focus on it (still using 4X objective). Lower the needle into the oil, bringing it into the same focal plane as the shard of cover glass. Raise the magnification by switching to the 40X objective. Refocus on the edge of the shard and reposition the tip of the needle, if necessary.

    7. To open up the tip of the injection needle, GENTLY nudge the needle against the edge of the cover glass shard, while at the same time applying a short pulse of gas via the foot pedal. The needle will be sufficiently broken open when small droplets of liquid escape the end of the needle. Excess liquid flow due to too large an opening is not desirable, as it will kill the animals.

    8. Remove the agar pad from the stage by raising the needle up, but do not change the X- or Y-axis position. Slide the stage out from underneath the needle, remove the agar pad and place it onto a dissecting microscope. Transfer a worm onto the pad, below the surface of the oil. If the worm wriggles, GENTLY stroke it until it contacts the agar pad. The moist worm should adhere to the dry agarose.

    9. Injection
      1. Quickly place the agar pad back onto the stage, centering the worm in the field of view.
      2. Lower the needle into the same plane of focus as the worm.
      3. Switch the filters to Hoffman Illumination (a type of contrast filter). This allows morphological detail of the worm to become visible. If Nomarski/DIC optics are available on the inverted scope that will work as well. Using the 40X objective, focus on the "grainy" syncytial center of the worm gonad,  below the "honeycomb" pattern of the germ nuclei (choose whichever gonad is positioned on the side of the worm facing the needle).
      4. Fine tune the position of the needle until the tip is also in focus (the same plane as the gonad "graininess").
      5. Gently insert the needle into the center of the gonad and apply a brief pulse of gas pressure to expel a droplet or two of DNA into the gonad arm. You should observe a wave of liquid spread across the distal gonad. Do not assume more liquid is better, since too much liquid can flow into the proximal bend of the gonad arm, which can shut down oocyte production. If possible, depending on the position of the worm, inject the other gonad arm in the same manner.
      6. It is important to work quickly while injecting a worm, as it can easily desiccate. The decision to inject the second gonad arm is dependent upon how quickly you can re-position the needle and worm. The entire process should ideally take less than 1-2 minutes.

    10. Remove the cover glass from the stage and place it on a dissecting scope. Apply 1µl of M9 buffer (22mM KH2PO4, 42mM Na2HPO4, 85mM NaCl, 1mM MgSO4) directly onto the injected worm (this might entail submerging the pipette tip below the surface of the halocarbon oil). The buffer should rehydrate the worm; it will then float off the agarose pad surface. Transfer the worm to a regular plate using a worm pick The agarose pad can be reused several more times until it has become too saturated with buffer and the worms no longer adhere.

    Transgenic selection

    1. Injected worms, the P0 generation, are transferred to individual fresh, bacterially seeded plates (60 mm) and allowed to reproduce. Typically, injected animals are incubated at 20°C for 2-3 days until the offspring appear.

    2. The F1 offspring are observed for evidence of the transgenic marker (such as fluorescence) using a fluorescent stereomicroscope. Each fluorescent, carrier F1, animal is separately cloned onto a new plate (since offspring from each F1 are considered a distinct line). The F1 hermaphrodites are allowed to reproduce. Again, this is typically performed at at 20°C for 2-3 days until the offspring appear.

    3. The F2 generation is observed for transgenic animals as well. F2 animals that have inherited and express the transgenic array are considered a "stable" line.

      NOTE: At the F1 stage there may be many transgenic animals, but they are not stable. The majority of F1 transgenic animals will not be propagated into F2 stable lines.

      NOTE: To control for the variability in the gene copy number, generate a minimum of three independent stable lines per construct injected.

    4. Each independent stable line should be maintained as a separate line of worms. Each line can be propagated by transferring several transgenic animals to a new plate at each generation; this must be performed using a fluorescent stereomicroscope if the selectable marker is fluorescent.

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    When doing this procedure, it is important to remember to:
    - inject directly into the center of the gonad
    - not inject too much liquid
    - work quickly to prevent dessication.

    If you run into problems with the needle, such as it is broken too large, it is best to remake a fresh needle, rather than trying to inject with a less than ideal needle.

    The generation of transgenic worms has many applications such as:
    - identification of mutant genes, in method called transformation rescue
    - mapping of protein domains through expression of truncated proteins
    - characterization of the role of a gene in cellular and disease processes.

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    Acknowledgements

    We wish to acknowledge the cooperative spirit of all Caldwell Lab members. Movement disorders research in the lab has been supported by the Bachmann-Strauss Dystonia & Parkinson Foundation, United Parkinson Foundation, American Parkinson Disease Association, Parkinson's Disease Association of Alabama, the Michael J. Fox Foundation for Parkinson's Research, and an Undergraduate Research.

    Materials

    Name Type Company Catalog Number Comments
    Agarose Ultrapure Invitrogen 15510-027
    Halocarbon Oil, Voltalef Hulle 10S elfatochem, France
    Coverglass 18x18 mm Fisher Scientific 12-548-A
    Coverglass 20x30 mm Fisher Scientific 12-548-5A
    Coverglass 22x50 mm Fisher Scientific 12-545-E
    100 ul Capillary VWR international 53432-921
    Glass Capillary for Needles "Kwik-Fil" World Precision Instruments, Inc. 1B100F-4
    Needle Puller Tool Narishige International Model PP-830
    microINJECTOR System Tritech Research, Inc. MINJ-1000 Scope, Stage, Manipulator
    Dissecting, with Fluorescence Microscope Nikon Instruments SMZ800
    Dissecting Microscope Nikon Instruments SMZ645

    References

    1. Stinchcomb, D.T., Shaw, J.E., Carr, S.H. and Hirsh, D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484-3496 (1985).

    2. Mello, C.C., Kramer, J.M., Stinchcomb, D., and Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970 (1991).

    3. Kelly, W. G., Xu, S., Montgomery, M.K. and Fire, A. Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene. Genetics 146, 227-238 (1997).

    4. Cao, S., Gelwix, C.C., Caldwell, K.A., Caldwell, G.A. 2005. Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans. J Neurosci 25, 3801-3812 (2005).

    5. Caldwell, G. Integrated Genomics: A Discovery-Based Laboratory Course. : John Wiley and Sons. West Sussex, England, 2006.

    6. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77: 71-94 (1974).

    Comments

    11 Comments

    Dear Dr. Laura A,   I am very much interested to work with you as a Researcher. Regarding myself, I would like to inform you that I did my Ph.D. degree from the Faculty of Science, Hiroshima University, Japan. I completed my M.Phil. M.Sc. and B.Sc. (Honours) degree from the University of Rajshahi, Bangladesh. Now I am working on toxic response and behavior of C. elegans as a postdoctoral researcher at Pusan National University, S. Korea.    I would be glad if you would kindly accept me as a Researcher in your Laboratory and provide me a fellowship or any financial support.   I look forward to your kind reply at your earliest convenience.   With best regards   Sincerely yours, Dr. M. Golam Mortuza Present Address Postdoctoral Researcher Lab of Ecology and Behavior System Division of Biological Sciences Pusan National University Busan 609-735, S. Korea E-mail: mortuzaru@yahoo.com   Permanent Address Professor Department of Zoology Rajshahi University Rajshahi 6²05, BANGLADESH E-mail: mortuza@ru.ac.bd
    Reply

    Posted by: AnonymousApril 15, 2009, 2:41 AM

    Hi, i am learning microinjection in C. elegans and am having problems recovering the worms.  They seem to recover fine initially and are thrashing around in the buffer on the plate, but after a day when I look back they have all died on the plate where they were placed.  I don't know what is wrong, do you have any suggestions? Thanks. Jane
    Reply

    Posted by: AnonymousMay 22, 2009, 1:57 PM

    Dear Jane,
    Death post injection is likely from two causes. The first is that the worms spent too much time stuck down on the agar pad before being hydrated off. Often they will be alive (barely) but then die. Reducing the time they spend stuck down will stop this source of death. As you become more adept and quicker with the process this will improve. The second cause of post injection death is from the worm getting stabbed too much- either from being injected in the wrong place (intestine), being injected too deeply (the &#x²01C;shish-kabob&#x²01D; mistake) or using too big of a needle. Often one can tell if this is the problem when the next day the injected worm has exploded out its guts. Make sure you are using a very tiny, fine tip in the needle and only barely enter under the cuticle into the gonad. Again these will improve with practice.

    One last thought- when you transfer the worm from the injection pad to the plate sometimes oil comes along with it. Gently move the worm out from the oil droplet onto the food. This will help it recover.
    Good luck!

    Laura
    Reply

    Posted by: AnonymousMay 26, 2009, 1:18 PM

    Hello!

    I am currently designing an automated system for c.elegans injections that involves circulating worms through microchannel in a PDMS chip and positioning them for injection from a microneedle. One of the important aspects of our design is to fabricate microneedles which can more easily puncture the worm's cuticle due to smaller needle size.

    However, we have been unable to find any reference, print or electronic, that has measured the force needed to puncture the worm. This force is important in choosing how thin our needles can be, and of course the force dŒs vary with needle tip size. We may need to experimentally determine this "puncture strength" ourselves as the first to ever do so, but I am wondering if you might know of anyone who has measured similar parameters that we can at least get a ballpark place to start.

    Thanks so much!

    -- Andrew
    Reply

    Posted by: AnonymousNovember 22, 2009, 5:52 PM

    Andrew,

    We are not aware of the type of precise information on puncture strength or penetration forces required for C. elegans, as we are molecular biologists, but your question reminded me of a method I remembered (and once tried without success) from the late 1990s. The technology you describe is likely much improved or can be. Check out this paper and maybe contact the authors regarding your question.

    Genetic transformation of nematodes using arrays of micromechanical piercing structures.
    Hashmi S, Ling P, Hashmi G, Reed M, Gaugler R, Trimmer W.
    Biotechniques. 1995 Nov;19(5):766-70.

    Best of luck with your experiments. If you are ever interested in us to beta-test something as collaborators, please let us know.

    Guy
    Reply

    Posted by: AnonymousNovember 22, 2009, 8:38 PM

    Hello,

    I am currently trying to inject double stranded DNA as an alternative approach to do tissue specific knockdown. I am following a protocol in which DNA sense and antisense fused to tissue specific promoter are injected together with a marker. According to the protocol, I should inject the PCR product directly and at the concentration of 100 ng/ul. However, when I injected this mixture into the gonad, most of the worms don't survive more than one day, which is not the case with the normal injection for generating the transgenic line.

    DŒs someone have any experience with this and can suggest me something that I should change or incorporate in my protocol to make the injection work?

    Thanks before,
    Yemima
    Reply

    Posted by: AnonymousJune 6, 2011, 12:21 PM

    We would like to start doing microinjections in our lab and I am would like to know where you acquired the halocarbon oil. In the Wormbook microinjection protocol, Series 700 Halocarbon oil from Halocarbon Products in NJ was used. Do you know how this compares to the Voltalef halocarbon oil that you used?

    Thank you very much for your help and for the wonderful instructional video!

    -Erin
    Reply

    Posted by: Erin M.March 13, 2013, 1:40 PM

    Dear Erin,
    We no longer can get the Voltalef oil and have too switched to using Halocarbon oil 700 (but ours is from Sigma). It is a bit thinner than the Voltalef, but it works as well, if not even a little better. Glad you like the video. Good luck with the injections. --Laura
    Reply

    Posted by: Laura B.March 13, 2013, 2:59 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter