Funding for our work is provided by the NIH, the March of Dimes, and the American Cancer Society.

Modified versions of this transgenesis approach were initially described in 1 and 2.
A. Sperm nuclei preparation
This nuclei preparation method is adapted from Murray 3, but the protease inhibitors have been omitted as they interfere with subsequent development of eggs transplanted with sperm nuclei. Aliquots are frozen at -80&deg;C and can be used for transplantations for approximately 6 months.
All solu...

<ol>
	<li>Kroll, K. L., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Xenopus+embryos+from+sperm+nuclear+transplantations+reveal+FGF+signaling+requirements+during+gastrulation.">Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.</a> Development. 122, 3173-3183 (1996).</li><li>Amaya, E., Kroll, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+generating+transgenic+frog+embryos.">A method for generating transgenic frog embryos.</a> Methods Mol Biol. 97, 393-414 (1999).</li><li>Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+extracts.">Cell cycle extracts.</a> Methods Cell Biol. 36, 581-605 (1991).</li><li>Hartley, K. O., Hardcastle, Z., Friday, R. V., Amaya, E., Papalopulu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Xenopus+embryos+reveal+that+anterior+neural+development+requires+continued+suppression+of+BMP+signaling+after+gastrulation.">Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.</a> Developmental Biology. 238, 168-184 (2001).</li><li>Karaulanov, E., Kn&#246;chel, W., Niehrs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+BMP4+synexpression+in+transgenic+Xenopus.">Transcriptional regulation of BMP4 synexpression in transgenic Xenopus.</a> EMBO J. 23, 844-856 (2004).</li><li>Ogino, H., Fisher, M., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergence+of+a+head-field+selector+Otx2+and+Notch+signaling:+a+mechanism+for+lens+specification.">Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification.</a> Development. 135, 249-2458 (2008).</li><li>Taylor, J. J., Wang, T., Kroll, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tcf-+and+Vent-binding+sites+regulate+neural-specific+geminin+expression+in+the+gastrula+embryo.">Tcf- and Vent-binding sites regulate neural-specific geminin expression in the gastrula embryo.</a> Developmental Biology. 289, 494-506 (2006).</li><li>Marsh-Armstrong, N., Huang, H., Berry, D. L., Brown, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+transgenes+in+Xenopus+laevis.">Germ-line transmission of transgenes in Xenopus laevis.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 14389-14393 (1999).</li><li>Offield, M. F., Hirsch, N., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+Xenopus+tropicalis+transgenic+lines+and+their+use+in+studying+lens+developmental+timing+in+living+embryos.">The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos.</a> Development. 127, 1789-1797 (2000).</li></ol>

For each transgenic construct to be tested, we generally transplant nuclei into 500-1000 eggs; at this scale, we can generate transgenic embryos expressing up to 10 different constructs per day, depending on how many females are induced to lay eggs. Of these transplantations, about one third of the eggs cleave and 60-80% of these cleaving embryos proceed through gastrulation normally. Depending on the reaction conditions used, between 10-50% of these embryos express the transgene of interest. Therefore, once this procedu...

A.Sperm nuclei preparation
Reagents:
<ol>
 <li>1X MMR (2mM CaCl2, 5mM HEPES, pH7.5, 2mM KCl, 1mM MgCl2, 100mM NaCl).</li>
 <li>0.1% Tricaine Methanesulfonate (MS222, aminobenzoic acid ethyl ester, Sigma A-5040), 0.1% sodium bicarbonate. Dissolve in water.</li>
 <li>2X Nuclear Preparation Butter (NPB). On the day of the sperm nuclei preparation, make up 30 ml of 2X NPB from aliquots of the stock solutions stored frozen: 500 mM sucrose (1.5 M stock), 30 mM HEPES (1M stock; titrate with KOH so that pH 7.7 is at 15 mM), 1 mM spermidine trihydrochloride (Sigma S-2501; 10 mM stock), 0.4 mM spermine tetrahydrochloride (Sigma S-1141; 10 mM stock), 2 mM dithiothreitol (Sigma D-0632; 100 mM stock), 2 mM EDTA (500 mM EDTA, pH 8.0).</li>
 <li>Use the 2XNPB to make a. 30ml 1X NPB, b. 10ml 1XNPB+3%BSA (fraction V, Sigma A-7906), c. 5ml 1XNPB+0.3%BSA.</li>
 <li>Lysolecithin: 100 &mu;l of 10 mg/ml L-&alpha;-lyso-Lecithin, Egg Yolk (Calbiochem, 440154); dissolve at room temperature just before use. Store solid stock at 20&deg;C. Discard the stock powder if it becomes sticky.</li>
 <li>Bovine serum albumin (BSA): 10% (w/v) BSA (fraction V, Sigma A-7906) Make up 5 ml in water on the day of the sperm nuclei preparation.</li>
 <li>Sperm storage buffer (1ml) 1X NPB, 30% glycerol, 0.3% BSA.</li>
 <li>Sperm dilution buffer: 250 mM sucrose, 75 mM KCl, 0.5 mM spermidine trihydrochloride, 0.2 mM spermine tetrahydrochloride. Titrate to pH 7.3-7.5 and store 0.5-1 ml aliquots at 20&deg;C.</li>
 <li>Hoechst No. 33342 (Sigma B-2261): 10 mg/ml stock in dH2O, store in a light tight vessel at 20&deg;C.</li>
</ol>
Equipment:
<ul>
<li>Swinging bucket rotor and centrifuge</li>
<li>cheesecloth</li>
<li>dissection tools (forceps and scissors)</li>
<li>fluorescence microscope</li>
<li>funnel</li>
<li>gloves</li>
<li>hemocytometer</li>
<li>needles (26 gauge)</li>
<li>paper towels</li>
<li>petri dishes (60 mm)</li>
<li>pipettes</li>
<li>plastic (5 and 10 ml)</li>
<li>Pipetman tips (1 ml and 200&mu;l)</li>
<li>Syringes (1 ml)</li>
<li>tubes (14 ml; Falcon, 2059)</li>
<li>tubes</li>
<li>microcentrifuge (1.5 ml)</li>
</ul>
B. Preparation of High Speed Extract
 Reagents:
 <ol>
 <li>1X Marc's Modified Ringer (MMR): 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.5. Prepare a 10X stock, and adjust pH with NaOH to 7.5.</li>
 <li>20X Extract buffer (XB) salt stock: 2 M KCl, 20 mM MgCl2, 2 mM CaCl2, filter-sterilize and store at 4&deg;C.</li>
 <li>Extract buffer (XB; freshly prepared and stored on ice): 1X XB salts, 50 mM sucrose,10mM HEPES (1 M stock, titrated with KOH so that pH is 7.7 when diluted to 15 mM; filter-sterilize, and store in aliquots at 20&deg;C). Prepare about 100 ml.</li>
 <li>2% (w/v) L-Cysteine hydrochloride 1-hydrate: Made up in 1X XB salts before use and titrated to pH 7.8 with NaOH. Prepare about 300 ml.</li>
 <li>CSF-XB: 1X XB salts, 1 mM MgCl2 (in addition to MgCl2 present in XB salts; final concentration 2 mM), 10 mM HEPES, pH 7.7, 50 mM sucrose, 5 mM EGTA, pH 7.7. Prepare 50 ml.</li>
 <li>Protease inhibitors: Mixture of leupeptin, chymostatin, and pepstatin, each dissolved to a final concentration of 10 mg/ml in dimethyl sulfoxide (DMSO). Store in small aliquots at 20&deg;C.</li>
 <li>1 M CaCl2.</li>
 <li>Energy mix: 150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl2.</li>
 <li>Pregnant Mare Serum Gonadotropin (PMSG): 100 U/ml PMSG (P.G.600&reg;, Intervet, Inc., 021825). Dissolve in water and stored at 20&deg;C.</li>
 <li>Human Chorionic Gonadotropin (HCG): 1000 U/ml HCG (CHORULON&reg;, Intervet, Inc., 057176 ). Dissolve in water and stored at 4&deg;C.</li>
 </ol>
 Equipment:
 <ul>
 <li>Xenopus laevis females</li>
 <li>Needles (18 and 26 gauge)</li>
 <li>Pasteur pipette</li>
 <li>wide bore</li>
 <li>Syringes (1 mL)</li>
 <li>Tubes, microcentrifuge (0.5 mL)</li>
 <li>Tubes, thick-wall polycarbonate (Beckman, 349622)</li>
 <li>Tubes, ultraclear (14 x 95 mm; Beckman, 344060)</li>
 <li>Ultracentrifuge and rotors (e.g., Beckman TL-100 with rotors SW 40 Ti and TLA-100.3)</li>
 <li>Beakers for egg collection</li>
 <li>Buckets or containers for holding female frogs (e.g., 4-L plastic beakers with mesh lids).</li>
 </ul>
 C. Nuclear transplantation.
 Reagents:
 <ol>
 <li>2.5% agarose in 0.1XMMR (for making injection dishes)</li>
 <li>2.5% Cysteine in 1XMMR, pH8.0, prepared on the day of use</li>
 <li>Ficoll</li>
 <li>10 mg/ml gentamycin (1000X stock)</li>
 <li>high speed egg extract (see above)</li>
 <li>100 MgCl2</li>
 <li>10X MMR (see above)</li>
 <li>Restriction enzyme (e.g. NotI from New England Biolabs)</li>
 <li>Sperm dilution buffer (SDB; see above) and sperm nuclei (see above)</li>
 <li>Human Chorionic Gonadotropin (HCG) as above</li>
 <li>mineral oil (Sigma, M8410)</li>
 <li>Linearized plasmid to be introduced as the transgene: Prepare linearized plasmid at a concentration of about 100 ng/&mu;l in sterile, nuclease-free water (we avoid Tris and EDTA-containing buffers, which are somewhat toxic to embryos). The restriction enzyme used to linearize the plasmid does not have to be the same as the one used in the nuclear transfer reaction. We usually use NotI for all reactions, regardless of what plasmid is linearized with. Some calibration of the enzyme dilution used in the reaction may be necessary, as too much enzyme can cause adverse effects on post-gastrula development. Plasmid can be purified in several different ways: we usually use the Qiagen Qiaquick PCR purification kit according to the manufacturers directions; purification of a single band from a gel is not necessary. If plasmid is purified using phenol/chloroform extractions and ethanol precipitation, be certain to remove all traces of organics and ethanol.</li>
 </ol>
 Equipment:
 Agarose dishes for injection: In a 60 mm plastic petri dish, lay a small 35mmX35mm weigh boat on molten 2.5% agarose in water 0.1XMMR to create a depression with an agarose-coated bottom for filling with eggs. Once agarose has hardened, wrap in parafilm and store at 4&deg;C until use. Make 2-3 dishes in advance for each transgenic reaction you plan to do.
 Infusion pump: We use a single syringe infusion pump from Harvard Apparatus, equipped with a 3 cc syringe/needle filled with mineral oil (Sigma M-8410). Blunt the syringe needle tip (to keep it from perforating the tubing) and attach the fine tygon tubing. Run the pump at ~10nl/sec; this assumes that the time the needle is in each egg will be no greater than 1 sec. Pump should be pre-run for several minutes prior to starting transgenesis for the day to assure that the plunger for the syringe is flush with the piston and that steady positive flow of oil out of the tubing is occurring.
 Needles for nuclear transfers. Using a micropipette puller, generate needles with long, sloping tips. Clip these with a forcep under a dissecting microscope equipped with an ocular micrometer to obtain an ~80 micron opening with a beveled shape.
 Other equipment: Xenopus laevis females, stereomicroscope, incubator, micromanipulator, microinjection needle puller (e.g. Model P-87, Sutter), syringe needles (26 gauge), glass microinjection needles, ocular micrometer for calibrated clipping of microinjection needle tips to 80&mu;m diameter, petri dishes, weigh boats 35mm, Tygon tubing (ID=1/32 in., OD=3/32 in.)

production of transgenic xenopus laevis by restriction enzyme mediated integration and nuclear transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Watch this Scientific Journal Video about Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation at JoVE.com

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

production-transgenic-xenopus-laevis-restriction-enzyme-mediated

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11.8K visualizações.  University of Manchester. Este protocolo de vídeo demonstra um método para a geração de transgênicos Xenopus laevis Por introdução de transgenes em núcleos do esperma seguida de transplante nuclear em ovos não fertilizados.