Making Transgenic Seamonkeys
Cathal Garvey
EXAMPLES
Example 1
PRODUCTION OF BRINE SHRIMP EGGS OR CYSTS
[0163] Adult female Brine shrimp are mechanically separated based on the visual identification of morphological differences between the male and female shrimp. Once the female shrimp are identified, they are transferred into a separate container and cultured in solution that contains 2-15% of NaCl, 1 to 10 ppm K 2 HP 0 4 . NaHCO 3 is used to adjust the solution pH to 8.0-9.0. The temperature of the culture is maintained at 20 to 30° C., and continually aerated using an aqua air pump, with an oxygen probe used to monitor the oxygen level. The Brine shrimp are fed with bacteria, yeast, microalgea, wheat flavor, soybean flavor, whey or even fresh chicken manure. After the culture reaches a density of between 500 to 5000 shrimps per liter, NaCl can be added to adjust the saline concentration to higher than 6.5% or the oxygen level can be reduced to below to 2 mg per liter. These changes to the culture conditions stimulate the female Brine shrimp to produce dormant eggs of cysts. Using a high saline concentration has the added advantage in causing the cysts to float on the surface of the culture, allowing their easy harvest using a fine net. Alternatively, the female Brine shrimp can be stimulated to produce cysts or eggs via the reduction of their food supply. The cysts or eggs can then be air-dried and stored for future use.
Example 2
[0164] Preparation of Decapsulated Eggs or Cysts for Gene Transformation and Hatching
[0165] The best way to introduce heterologous nucleic acids into Brine shrimp, is to start from a Brine shrimp egg or cyst. The eggs or cysts are surrounded by a very hard shell and have to be decapsulated before used for gene transformation. Dried cysts or eggs are first hydrated into a spherical shape with distilled water or sea water at room temperature for 30 to 90 minutes, longer incubation times are not recommended as the cysts or eggs will have started to resume their metabolisms therefore likely would not survive the decapsulation procedure. The decapasultion process uses the following steps: re-suspending one gram of eggs or cysts in 4.67 ml of water or sea water, followed by the slow addition of 0.33 ml of 10 N sodium hydroxide (40% NaOH). The solution must be stirred well and the reaction starts by the addition of 10 mls of house bleach (Clorox with 6% sodium hypochlorite). The solution should be kept at a temperature range of 20 to 30° C. The resulting mixture is stirred until the color of solution turns from dark brown to gray, to white and then to bright orange. The whole process usually takes less than 5 minutes, and is somewhat dependent on the mixtures temperature and the eggs and cysts themselves. Once the color change has occurred, immediately filter the solution through nets and rinse the eggs or cysts with fresh water to remove the chlorine. The eggs and cysts are washed in 0.1% sodium thiosulfate and 0.5% acetic acid solution for one minute to neutralize the reaction and completely remove the chlorine from the solution. The cysts are then re-washed well with fresh or salt water. The resulted decapsulated eggs and cysts can be used immediately for gene transfer or can be stored in a refrigerator for up to week for hatching.
Example 3
[0166] Gene Transfer into Brine Shrimp Eggs or Cysts
[0167] This example describes the process by which heterologous genes are expressed in the Brine shrimp eggs and cysts isolated in Example 1. A linear gene expression cassette, containing a eukaryotic promoter, a desired gene sequence, a polyA sequence and a transcription terminator is isolated by digestion with proper restriction enzyme from a cloned vector that contains the expression cassette. The desired DNA is then purified from agarose using either electrophoresis and/or column purification. Purified DNA is dissolved in pure water at suitable a concentration. A Bio-Red Gene Pulser is used in the present invention in order to introduce the DNA of interest into the Brine shrimp cyst. In order to perform this, 500 to 1000 cysts are re-suspend in 5 ml of autoclaved or sterilized sea water in a 9 mm petri dish and the appropriate amount of DNA is added, followed by incubation at 4° C. for 30 minutes. Approximately 0.8 mls of the cyst mixture is transferred to the Gene Pulser Cuvette. With a 0.8 ml volume. and 0.4 cm distance, electroportation parameters with Capacitance Extender are set at the voltage of 200-400 V and capacity at 125 to 960 μF. After electroporation, the cysts are incubated at room temperature in a petri-dish and placed on a slow rotation shaker. Following 24 to 36 hours of incubation, the Brine shrimp will hatch and the hatched Brine shrimp can be transferred into a container containing 100 to 500 ml seawater with aeration. The Brine shrimp are grown under the appropriate conditions and at around 12 to 14 days post-culture, single female Brine shrimp are separated into individual containers and grown under optimal conditions to allow them to reproduce via parthenogenesis. The transduced Brine shrimps are analyzed with maker gene expression, gene product immune detection, PCR or any other gene analysis methods.
Example 6
[0177] Isolation and Purification of a Transgenic Protein from a Brine Shrimp Culture
[0178] The transgenic Brine shrimp is harvested, washed with cold fresh water several times and homogenized in cold extraction buffer (e.g. 10 mM Tris, pH 6.5 to 8.5, 20 to 500 mM NaCl, 0.01 to 100 mM EDTA, 0.001 to 1% Triton X100, protease inhibitors). The solution is centrifuged and the pellet is solubilized with either 8 M urea or 6 M guanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. The solubilized protein is added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 min to 1 hour at room temperature with continuous agitation. After incubation, the resin and protein mixture is poured through a disposable column and the flow through is collected. The column is washed with 10-20 column volumes of the solubilization buffer. The protein is eluted from the column using 8 M urea, 10 mM tris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. A SDS-PAGE gel is run to determine which fractions to pool for further purification. As a final purification step, a strong anion exchange resin such as Hi-Prep Q (Biorad) is equilibrated with the appropriate buffer and the pooled fractions from above are loaded onto the column. Each protein is eluted off of the column with an increasing salt gradient. Fractions are collected as the column is run and another SDS-PAGE gel is run to determine which fractions from the column to pool. The pooled fractions are dialyzed against 10 mM Tris pH 8.0. This material is then evaluated for acceptable purity as determined by SDS-PAGE or HPLC, concentration as determined by Lowry assay or Amino Acid Analysis, identity as determined by amino terminal protein sequence, and endotoxin level as determined by the Limulus (LAL) assay. The protein is frozen after filtration through a 0.22 micron filter.
[0179] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.
[0180] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
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Nathan McCorkle
2010/11/14 Cathal Garvey <cathal...@gmail.com>:
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
kingjacob
Jacob Shiach
Andrew Barney
LOL.
2010/11/15 kingjacob <king...@gmail.com>:
ntchemod
On Nov 15, 10:12 am, Andrew Barney <keen...@gmail.com> wrote:
> haha, Every home should have multicolored transgenic seamonkeys!
>
> LOL.
>
> > Fan, Hao
> > http://www.freepatentsonline.com/y2004/0194156.html
>
> > On Mon, Nov 15, 2010 at 10:34 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> >> reference to the author?
>
>
> read more »
Cathal Garvey
haha, Every home should have multicolored transgenic seamonkeys!
LOL.
Cathal Garvey
Cathal Garvey
Andrew Barney
I was wondering how to get a needle that small... Now i know.. Very Helpful.
Nib
not stable.
it is likely that any nonstable transgenic artemia will lose its new
dna after some generations.
Do you know the mechanism by which this linearized poly-A dna piece is
integrated into the genome? Homologous recombination?
On 15 Nov., 21:40, Andrew Barney <keen...@gmail.com> wrote:
> Wow, thanks for that protocol Cathal!
>
> I was wondering how to get a needle that small... Now i know.. Very Helpful.
>
> >http://www.ceolas.org/VL/fly/protocols.html
> > Here's more drosophila protocols, many will be applicable to Artemia.
> > By the way, folks who wanna do DIYbio stuff: Here's your chance to pave new
> > ground in biotech. I couldn't find much info on Artemia biotech, so by
> > buying a Sea Monkey kit you can start generating data right now!
> > I suggest a hashtag if someone's thinking of doing this: #SciMonkeys
>
>
> >> I figure methods that are applicable to Drosophila have higher odds of
> >> working well with Artemia, and there's an excellent protocol for embryo
> >> transformation in Drosophila here:
> >>http://groups.molbiosci.northwestern.edu/carthew/manual/Injection.html
> >> It covers everything, from making the microneedle (by immersion of a
> >> stretched capillary pipette in sterilised, magnetically stirred grinding
> >> slurry!), to removing the coat without using bleach (they abrade the egg
> >> coat, it seems), to the microinjection process.
> >> With these techniques, which should be directly applicable to Artemia, you
> >> just need a genetic construct that can stably integrate and express
> >> luciferase in times of plenty, or after a certain life stage.
>
Cathal Garvey
I think the idea is to catch them at the single cell stage and overwhelm them with so much DNA that some will just *have* to integrate somewhere by accident. Only integrants that get it somewhere moderately safe will survive to adulthood.
Embryos often have active homologous recombination systems but I don't know if that applies here.
Often, it's a transposon you use to get things stably integrated. I gather efficiency is pretty good. The problem with transposons is, they may be unstable later on.
Is the Artemia genome sequenced?
Sent from my Android.
On 15 Nov 2010 21:24, "Nib" <ruedi...@googlemail.com> wrote:
I think this transfection will only produce transient expression, but
not stable.
it is likely that any nonstable transgenic artemia will lose its new
dna after some generations.
Do you know the mechanism by which this linearized poly-A dna piece is
integrated into the genome? Homologous recombination?
On 15 Nov., 21:40, Andrew Barney <keen...@gmail.com> wrote:
> Wow, thanks for that protocol Cathal!...
> On Mon, Nov 15, 2010 at 12:04 PM, Cathal Garvey <cathalgar...@gmail.com> wrote:
> >http://www.ceol...
> > On 15 November 2010 19:02, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
> >> I figure methods...
> >> On 15 November 2010 18:48, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
> >>> My thoughts ex...
> >>> On 15 November 2010 18:12, Andrew Barney <keen...@gmail.com> wrote:
>
> >>>> haha, Every home ...
Cory Tobin
Nope, but someone is working on it: http://www.mgel.msstate.edu/artemia.htm
-Cory
rwst
>
> Nope, but someone is working on it: http://www.mgel.msstate.edu/artemia.htm
http://www.uniprot.org/uniprot/?query=taxonomy%3Aartemia&sort=score
There appears to be ten species, the sequences are from
A. salina and A. sanfranciscana. Also dozens of named
varieties, according to
http://www.uniprot.org/taxonomy/6660
The closest already sequenced genome is that from insects,
as I cannot find even one crustacean fully sequenced.
rwst
Tristan Eversole
Also, this seems relevant: Recent advances in crustacean genomics. Integr. Comp. Biol. (2008) 48(6): 852-868 first published online October 30, 2008 doi:10.1093/icb/icn096
They have the full text (yay!): http://icb.oxfordjournals.org/content/48/6/852.full
I've only skimmed it, though. It is truly heartbreaking to see how little attention has been paid to the Crustacea.
People are talking about the amphipod Parhayle hawaiensis as a model organism for evo-devo, so I assume it'll get sequenced eventually. It doesn't look too hard to keep alive, either: http://www.extavourlab.com/protocols/Parhyale%20hawaiensis%20culture.pdf . Definitely not as easy as brine shrimp, but not too bad.
.....I always wanted to sequence hermit crabs. No, there is no good reason for this sentiment at all. I just think they're cute.