Name: Generation of Stable Transgenic C. elegans Using Microinjection (Video) | JoVE Journal
Uploaded: 2008-08-15T12:12:00.000+00:00
Duration: 12 min 9 s
Description: Learn microinjection techniques to generate stable transgenic C. elegans for visualizing gene expression in vivo. This method enables researchers to study genetic mechanisms using fluorescent microscopy.

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5 minutes

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 &amp; 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.

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
<ol>
<li>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. </li>
<li>The cDNA of the gene of interest can be cloned in two ways: <ol>
<li>Conventional cloning using restriction enzymes.</li>
<li>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.</li>
</ol></li>
</ol>
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
<ol>
<li>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/&micro;l each. </li>
<li>Prepare agar pads: <ol>
<li>Make a 2% agarose solution in water; heat or microwave until dissolved.</li>
<li>Set out several 22 x 50 mm cover glasses on a benchtop.</li>
<li>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.</li>
<li>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).</li>
<li>Store the pads in a cover glass box at room temperature. </li>
</ol></li>
<li>Make needle loaders for DNA solution: Needle loaders are created from 100 &micro;l 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. </li>
<li>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. </li>
<li>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. </li>
<li>Grow wild type (N2) worms to the early adult stage for injection using standard procedures 6. Prepare about 100 properly staged worms/construct injected.</li>
</ol>
Procedure
<ol>
<li>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. </li>
<li>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 &micro;l). </li>
<li>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. </li>
<li>Insert the needle into the microinjection arm, being careful to place it within the small internal rubber gasket. </li>
<li>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. </li>
<li>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. </li>
<li>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. </li>
<li>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. </li>
<li>Injection <ol>
<li>Quickly place the agar pad back onto the stage, centering the worm in the field of view.</li>
<li>Lower the needle into the same plane of focus as the worm.</li>
<li>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,&nbsp; below the "honeycomb" pattern of the germ nuclei (choose whichever gonad is positioned on the side of the worm facing the needle).</li>
<li>Fine tune the position of the needle until the tip is also in focus (the same plane as the gonad "graininess").</li>
<li>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.</li>
<li>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. </li>
</ol></li>
<li>Remove the cover glass from the stage and place it on a dissecting scope. Apply 1&micro;l 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.</li>
</ol>
Transgenic selection
<ol>
<li>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&deg;C for 2-3 days until the offspring appear. </li>
<li>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&deg;C for 2-3 days until the offspring appear. </li>
<li>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. </li>
<li>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.</li>
</ol>

<ol><li>Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrachromosomal+DNA+transformation+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Mol.+Cell.+Biol%22%5BJournal%5D)">Extrachromosomal DNA transformation of Caenorhabditis elegans.</a> Mol. Cell. Biol. 5, 3484-3496 (1985).</li>
<li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+transfer+in+C.elegans%3A+extrachromosomal+maintenance+and+integration+of+transforming+sequences%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li>
<li>Kelly, W. G., Xu, S., Montgomery, M. K., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+requirements+for+somatic+and+germline+expression+of+a+generally+expressed+Caenorhabditis+elegans+gene%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene.</a> Genetics. 146, 227-238 (1997).</li>
<li>Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Torsin-mediated+neuroprotection+from+cellular+stresses+to+dopaminergic+neurons+of+C.+elegans%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans.</a> J Neurosci. 25, 3801-3812 (2005).</li>
<li>Caldwell, G. Integrated Genomics: A Discovery-Based Laboratory Course. , John Wiley and Sons. West. Sussex, England. (2006).</li>
<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetics+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li>
</ol>

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 ...

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

generation of stable transgenic c. elegans using microinjection

Generation of Stable Transgenic C. elegans Using Microinjection

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.

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

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Sea elegance is an incredibly useful model organism due to both our knowledge of its cell lineages as well as the wealth of information produced from having sequenced and analyzed its entire genome. In this video, we demonstrate the process of micro injecting plasmids into sea elegance and the selection of transgenic offspring using fluorescent microscopy as a transparent and multicellular animal, sea elegance is ideal for visualizing transgenes expressing fluorescently labeled proteins in vivo. Hi, my name is Laura Berkowitz and I'm a research associate in the Caldwell Lab in the Department of Biological Sciences here at the University of Alabama.And I'm Adam Knight, also from the Calwell Lab at the University of Alabama. Today I will be showing you how to prepare for and perform Plasmin Microinjection to generate transgenic sea elegance, And I'll be showing you how to select and maintain transgenic seal elegance lines. So let's get started.Before we begin the microinjection, we have a few preparation steps, which I'll show you now. First, prepare the plasmid DNA to be injected, two plasmids are required, one that encodes the tissue specific expression of our gene of interest, and the second that provides a selectable transformation marker. Mix the plasmids together in a one-to-one ratio of 50 nanograms per microliter.Each next, prepare auger pads, which will be used to immobilize the worms for injection. To make the auger pads first set out several 22 by 50 millimeter cover glasses on the benchtop. Using a pasture pipette place a drop of melted 2%agros on one cover glass, and immediately place a second cover glass over the drop at a 90 degree angle to the first.Let the aero solidify for a few seconds and then remove the top cover glass. The diameter of the flattened disc of aero should be between 15 and 20 millimeters. Like a pet, allow the pad to air dry completely for several hours or overnight.Once they are dry, store the pads in a cover glass box at room temperature. Next, prepare the needle loaders for the DNA solution. To do this, take a hundred microliter capillary tube and heat it in the middle of an open flame and rapidly pull it to approximately twice its length.Once the capillary tube cools, snap the two halves apart. Stockpile 10 to 20 needle loaders for injection. You can store them upright in a micro centrifuge tube.Brack, be careful. The needle loader points are very sharp. Now make the micro injection needles.These needles are made from a special capillary tube that contains an internal glass filament. This filament serves as a wick for the DNA solution. To make the needles we use a Niki PP eight 30 Polar With a heat setting of 24.8, several needles are pulled at a time for storage.Multiple needles can be mounted on a strip of modeling clay. Small shards of cover glass will be used to break open the pulled needle tip. These shards are prepared by wrapping an 18 by 18 millimeter cover glass in a Kim wipe and gently applying pressure until it breaks into several pieces.Use shards that are approximately three by four millimeters in size. In addition to preparing the DNA and needles, prepare about 50 to 100 properly staged. Two wild type worms for each expression vector construct to be I injected.We are now ready to set up the microinjection microscope. To begin setting up the microscope, open the main valve on the helium tank that is connected to the microinjection needle arm and holder. Close the regulator to allow the gas to enter the line.Bringing the pressure to 35 PSI the gas can be released using a foot pedal. Next, insert a needle loader into a standard mouth pipetter tubing and drop a small amount of the plasmid mixture approximately one microliter. Now gently insert the loader all the way through the microinjection needle until resistance is felt.Expel the DNA solution by gently blowing and then remove the loader. Once the DNA is loaded, insert the needle into the microinjection arm, being careful to place it within the small internal rubber gasket and then clamp it to the arm of the manipulator. Next place a needle breaking shard of cover glass on one edge of an auger pad.Cover the shard as well as the whole surface of the auger pad with hallow carbon oil. Then place the auger pad on the stage of an inverted microscope. After the auger pad is in place, position the needle in the center of the viewing field using the Forex objective and bright field illumination, but do not yet lower it into the oil.Next position the inner edge of the shard of cover glass in the center, and then focus on it. 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 40 x objective, then refocus on the edge of the shard and reposition the tip of the needle if necessary.Now break Open 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. Once the needle is ready, we begin the microinjection. Next, remove the auger 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 auger pad and place it onto a dissecting microscope.Transfer a word onto the pad below the surface of the oil. If the worm wriggles gently stroke it until it contacts the auger pad, the moist worm should adhere to the dry agro. Quickly place the auger pad back onto the stage, centering the worm in the field of view.Then lower the needle into the same plane of focus as the worm. Switch the filters on the inverted scope to Hoffman illumination. This contrast filter allows morphological detail of the worm to become visible.Using the 40 x objective, focus on the grainy sensorial 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.Fine. Tune the position of the needle until the tip is also in focus.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 goad with a lot of practice and skill. You can hit the goad center every time.Keep in mind that it's important to work quickly while injecting a worm. As it can easily desiccate. The entire process should ideally take less than one to two minutes.Once the injection is completed, remove the auger pad from the stage and place it on a dissecting scope. Apply one microliter of M nine buffer directly onto the injected worm. This might entail submerging the pipette tip below the surface of the halo carb oil.The buffer should rehydrate the worm and it will then float off the aeros pad surface, transfer the worm to a regular plate using a worm pick. Now I will hand off the injected worms to Adam and he'll show us the selection and maintenance of transgenic lines. The injected worms are transferred to individual fresh seeded 60 millimeter plates and allowed to reproduce.These worms represent the P zero generation. Typically injected animals are incubated at 20 degrees Celsius for two to three days until the offspring appear. Once the offspring appear, we are ready to begin screening for transgenic worms.The F1 offspring are observed for evidence of the transgenic marker. In this case, the selectable marker is fluorescent, so we can screen using a fluorescent stereo microscope. Each fluorescent carrier F1 animal is separately cloned onto a new plate.The injected P zero may only produce one to five fluorescent offspring among all of its F1.Allow the F1 hermaphrodites to reproduce at 20 degrees for two to three days until the offspring appear. Once the F two generation is ready, they're observed for transgenic animals as well. F two animals that have inherited and expressed a transgenic marker are considered a stable line.Each independent stable line should be maintained as a separate line of worms propagate each line by transferring several transgenic animals to a new plate at each generation. This must be performed using fluorescent stereo microscope. If the selectable marker is fluorescent to control for the variability in the gene copy number, generate a minimum of three independent stable lines per construct injected.We've just shown you how to micro inject expression, vector constructs, and see elgan and to select for stable lines. So that's it. Thanks for watching and good luck with your experiments.

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Biology

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Learn microinjection techniques to generate stable transgenic C. elegans for visualizing gene expression in vivo. This method enables researchers to study genetic mechanisms using fluorescent microscopy.

Generation of Stable Transgenic C. elegans Using Microinjection

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