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~NEW SPLICER~ Volume 1.0 November 2010

In this issue Topic of the month: Which pet would you like to see our latest labs create (with 99.9% chance of something working, 5% said something is of use)? The Story of Polly the Dodo A beginners guide to animal cloning... New splicer compendium -Species cloned thus far New Splicer How it all started... How to make a Dodo... and much more! Don’t forget to Breathe

Don’t forget to smile


Introduction ~ This really needs one ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ First off you will all be delighted to hear I am crazy, crazy in my dedication to all things fictionally factual. Or what was and is is slightly askew and abnormal. Just like me... Also Crazy... In this it is my intention to resurrect the Dodo as so far as factually fictionally possible. Using mirth, smiles, science, folly, fact and fiction and Jam. The jam is most important as you will discover. This will be something you will just have to let go literally in and discover for yourself. Although next time I will need more user participation as I only have so many split personalities to make this a socially non-biased magazine. So I heart-warmingly welcome you to our magazine, which we all have toiled, lost DNA, grown DNA and at the end with a little Factual fiction have a little version 1.8 running around our homes. ~New Splicer~ D

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Do Not Ask... “Did you hear the story about the peacock? It’s a beautiful tail! Don’t forget to Breathe

Don’t forget to smile


~~~~~~~~~ToPIC of the Month~~~~~~~~ The question posed to my readers and split selves was thus: Which pet would you like to see our latest labs create (with 99.9% chance of something working, 5% said something is of use)? I got back to myself pretty much straight away, twice in fact... However, I was alone in this endeavour... But it worked... We managed to create, to our delight, two new additions to the New Splicer list of available Pre-Extinct-Pet species...

New Splicer~ I choose Martya(Don’t)xwar

ORIGIN The manticore myth was of Persian origin, where its name was “man-eater” (from early Middle Persian ‫ایترام‬ martya”man” (as in human) and ‫ راوخ‬xwar- “to eat”).

Don’t forget to Breathe

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The manticore (Early Middle Persian Martyaxwar; Βάρἰκος Baricos in Greek) is a legendary creature similar to the Egyptian sphinx. It has the body of a red lion, a human head with three rows of sharp teeth (like a shark), and a trumpet-like voice. Other aspects of the creature vary from story to story. It may be horned, winged, or both. The tail is that of either a dragon or a scorpion, and it may shoot poisonous spines to either paralyze or kill its victims. It devours its prey whole. It leaves no clothes, bones, or possessions of the prey behind. During the early centuries, it was often believed that when a person would go missing, it was evidence that it was caused by a manticore and that manticore were real. The creature’s feet may be those of a dragon, but are most often described as the paws of a lion. Its size ranges from the size of a lion to the size of a horse. It is also mistaken as a bearded man when seen from a distance. This creature is wonderfully like me can’t make up its mind, can be anything and nothing... And is mistaken for a bearded man when seen from a distance! Amazing, I want one is what I said and New Splicer listened! So version 1.0 on its way preorders on self addressed envelope please... Question: What does a 1,000 lb. canary say? Answer: Here kitty, kitty, kitty! This is partly my inspiration and should be referenced: “And just when you thought it could get no stranger, May I present the semblance of a Scandanavian doppelganger. From the frozen depths of a forgotten fjord, The never sleeping for want of eating unholy stench of the manticore” ... Neil Fallon and Clutch Don’t forget to Breathe

Don’t forget to smile


How this came about I do not know, but several coincidences later and several Canadian friends told me they like this too: Ogopogo Ogopogo or Naitaka (Salish: n’ha-a-itk, “lake demon”) is the name given to a cryptid lake monster reported to live in Okanagan Lake, in British Columbia, Canada. Ogopogo has been allegedly seen by First Nations people since the 19th century. The most common description of Ogopogo is a forty- to fiftyfoot-long (12 to 15 m) sea serpent. It has supposedly been photographed and even been caught on tape. Well who needs tape when you have scientific insanity! That’s right, the Ogopogo is here for you delight and ours. ~New Splicer warning- does not mix well with other fish or humans~

Q. How do you catch a unique bird? A. Unique up on it. Q. How do you catch a tame bird. A. Tame way -- unique up on it. Don’t forget to Breathe

Don’t forget to smile


The Story of Polly the Dodo Polly, an Extinct Dodo, was the first Dodo to have been successfully cloned from an extinct renovated DNA. She was cloned at the Oregon National Dodo Research Center and the Oregon Stem Cell Center. She is doing well and appears happy with no signs of suicidal tendencies.Polly was publicly significant because the effort showed that the genetic material from ancient DNA, programmed to express only a distinct subset of its genes, can be re-derived to grow an entirely new organism. Before this demonstration, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg. However, this concept was not yet demonstrated in a extinct mammalian system.Cloning Polly the sheep had a low success rate per fertilized Emu egg; she was born after 237 eggs were used to create 29 embryos, which only produced three dodo’s at birth, only one of which changed its extinct status and survived. Seventy dodo’s have been created and one third of them died young (in dodo tearms). There were early claims that Polly the Dodo had pathologies resembling accelerated aging. Scientists speculated that Polly’s aging was related to the shortening of telomeres, DNAprotein complexes that protect the end of linear chromosomes. However, other researchers, including Dr M. P. Mitalipov who led the team that successfully cloned Polly, argue that Polly’s early death due to respiratory infetion was unrelated to deficiencies with the cloning process. ~New Splicer~ What’s orange and sounds like a parrot? A carrot!

Don’t forget to Breathe

Don’t forget to smile


A beginners guide to animal cloning... What is Animal Cloning? Animal Cloning is the process by which an entire organism is reproduced from a single cell taken from the parent organism and in a genetically identical manner. This means the cloned animal is an exact duplicate in every way of its parent; it has the same exact DNA. Cloning happens quite frequently in nature. Asexual reproduction in certain organisms and the development of twins from a single fertilized egg are both instances of Cloning. With the advancement of biological technology, it is now possible to artificially recreate the process of Animal Cloning. Development of Animal Cloning in the Lab. Scientists have been attempting to clone animals for a very long time. Many of the early attempts came to nothing. The first fairly successful results in animal cloning were seen when tadpoles were cloned from frog embryonic cells. This was done by the process of nuclear transfer. The tadpoles so created did not survive to grown into mature frogs, but it was a major breakthrough nevertheless.

After this, using the process of nuclear transfer on embryonic cells, scientists managed to produce clones of mammals. Again the cloned animals did not live very long. The first successful instance of animal cloning was that of Dolly the Sheep, who not only lived but went on to reproduce herself and naturally. Dolly was created by Ian Wilmut and his team at the Roslyn Institute in Edinburgh, Scotland, in 1997. Unlike previous instances, she was not created out of a developing embryonic cell, but from a developed mammary gland cell taken from a full-grown sheep. Don’t forget to Breathe

Don’t forget to smile


Since then Scientists have been successful in producing a variety of other animals like rats, cats, horses, bullocks, pigs, deer, dodo etc. You can even clone human beings now and that has given rise to a whole new ethical debate. Is it okay to duplicate nature to this extent(~New Splicer Vote YES~)? What would that do to the fabric of our society? [surely it would make the fabric more comfortable?] The Process of Animal Cloning Initial attempts at artificially induced Animal Cloning were done using developing embryonic cells. The DNA extracted from an embryonic cell and implanted into an unfertilized egg, from which the existing nucleus had already been removed. The process of fertilization was simulated by giving an electric shock or by some chemical treatment method. The cells that developed from this artificially induced union were then implanted into host mothers. The cloned animal that resulted had a genetic make-up exactly identical to the genetic make-up of the original cell. Since Dolly, of course, it is now possible to create clones from non-embryonic cells.

Two duck hunters out on the marsh duck hunting. One says to the other, “we’re not having much luck today getting any ducks.” The other one says, “ maybe we’re not throwing the dog high enough.”

Don’t forget to Breathe

Don’t forget to smile


Ethics of Animal Cloning While most scientists consider the process of animal cloning as a major break through and see many beneficial possibilities in it, many people are uncomfortable with the idea, considering it to be ‘against nature’ and ethically damning, particularly in the instance of cloning human beings. The truth is that most of the general public are not aware of the exact details involved in cloning and as a result there are a lot of misconceptions about the entire matter. [I was going to say until now but that would be an entire misconception]. A Frenchman walks into a bar with a parrot on his shoulder. The bartender asks, “Where did you get that thing?” The parrot replies, “In France, there are millions of them!” Advertisment

For Sale One second hand dodo bassinet... Like new! For All your New Splicer Dodo related needs! ~Requires Dodo fertilised egg and DNA~ Quote “I’m A dodo” for a 10% discount...

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~New Splicer Cloning Compendium ~ Landmark experiments[clarification needed] in chronological order: Tadpole: (1952) Many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.[citation needed] Carp: (1963) In China, embryologist Tong Dizhou produced the world’s first cloned fish by inserting the DNA from a cell of a male carp into an egg from a female carp. He published the findings in a Chinese science journal. Mice: (1986) A mouse was the first mammal successfully cloned from an early embryonic cell. Soviet scientists Chaylakhyan, Veprencev, Sviridova, and Nikitin had the mouse “Masha” cloned. Research was published in the magazine “Biofizika” volume ХХХII, issue 5 of 1987.[clarification needed] Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep from a somatic cell in 1997. Rhesus Monkey: Tetra (January 2000) from embryo splitting[clarification needed] Gaur: (2001) was the first endangered species cloned. Cattle: Alpha and Beta (males, 2001) and (2005) Brazil Cat: CopyCat “CC” (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons Dog: Snuppy, a male Afghan hound was the first cloned dog (2005). Rat: Ralph, the first cloned rat (2003) Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone. Horse: Prometea, a Haflinger female born 28 May 2003, was the first horse clone. Dodo: Polly, the first extinct species revived on 23rd November 2007. Water Buffalo: Samrupa was the first cloned water buffalo. It was born on February 6, 2009, at India’s Karnal National Diary Research Institute but died five days later due to lung infection. Camel: (2009) Injaz, is the first cloned camel

Manticore: (2010/11) First Xmas Pet? Ogopogo: (2010/11) First xWar Pet.

What do you call a melted penguin? Grey... Don’t forget to Breathe

Don’t forget to smile


New Splicer ~ How it all started Welcome to the Frozen Ark Project “Saving the DNA and the viable cells of the world’s endangered animals” The mission of the Frozen Ark Project is to collect, preserve and store tissue, gametes, viable cells and DNA from endangered animals. The project focuses on the thousands of animals that are threatened with extinction. What birds spend all their time on their knees? Birds of prey!

Animal species are dying out at an unprecedented rate. The current round of extinctions is largely created by mankind, because of the increase in human populations and its effect on the planet’s ecosystems. Global warming is a major contributor to this destruction. Despite the best efforts of conservationists, thousands of extinctions are happening. This pattern is common across all animal groups, both vertebrate and invertebrate, and emphasises the importance of collecting the genetic materials and cells of endangered animals before they disappear. The loss of a species destroys the results of millions of years of evolution. If cells are preserved, invaluable information about the species is saved. The Frozen Ark Project is not a substitute for conservation, but a practical and timely backup of genetic material for the good of, future generations. Don’t forget to Breathe

Don’t forget to smile


Samples are taken from captive breeding programmes, zoos and wild populations. The Project is providing uniquely important scientific knowledge and a precious source of genetic material for conservation and research. We believe that no more animals should be allowed to approach extinction without such material being conserved. The Frozen Ark has developed into an international consortium of museums, zoos, aquaria and research laboratories, all committed to the long-term preservation of this material. http://www.frozenark.org/

Two statisticians went duck hunting. A mallard flew overhead and one statistician fired just to the right of the bird. The other statistician fired just to the left of the bird. They turned to each other in glee, and congratulated each other... “On average, he’s dead!”, they cried! The mallard continued his migration.

Don’t forget to Breathe

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How to make a Dodo! Materials and Methods Cell preparation and culture A primary cell line is isolated from a mature animal (such as a sheep). After isolated, cells are washed in Dulbecco minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin, and the cells are seeded into six-well culture plates following typical cell culture techniques. Following the first passage, cells are grown to confluence and frozen in DMEM-F12 supplemented with 20% FCS and 10% dimethyl sulfoxide. After thawing, cells are cultured in DMEM supplemented with 10% FCS and 1% (v/v) penicillin/streptomycin to approximately 80% confluence (passages 2–5). Approximately half of the cells are allocated to be treated with culture medium containing 15 µM roscovitine (for approximately 24 hours prior to nuclear transfer), and the remaining cells are cultured with medium supplemented with 0.5% FCS (for 72 hours prior to nuclear transfer). The roscovitine-treated cells are exposed to the inhibitor throughout the nuclear transfer process. Donor cells to be submitted for flow cytometry sorting are trypsinized, centrifuged, and resuspended in 1 mL of physiological buffered solution (PBS). Cells are first incubated with DNase-free RNase A for 30 min at 37°C, then with 1 mg/mL propidium iodide for 10 min at 25°C before being processed on the flow cytometer (Campbell, 1999).

~MEMO~ Dodo Version 3.2 recall - Harmful when looked at directly and if under the immediate sense of ridicule. This is no JOKE please do not joke. Don’t forget to Breathe

Don’t forget to smile


Oocyte preparation and nuclear transfer Recipient oocytes are washed and selected after they are removed from animal. Only oocytes that have a homogenous cytoplasm and at least three layers of cumulus cells are selected for in vitro maturation. In vitro maturation medium consisted of tissue culture medium (TCM 199) supplemented with 10% FCS, 50 µg/mL sodium pyruvate, 1% penicillin/streptomycin (v/v), 1 ng/mL recombinant insulinlike growth factor 1, 0.01 U/mL bovine luteinizing hormone (LH), and 0.01 U/ mL bovine follicle-stimulating hormone (FSH). Selected oocytes are placed in 0.5 mL of maturation medium overlaid with mineral oil (0.4 mL) and incubated for 16–18 hours at 37°C in 5% CO2 and air. After maturation, oocytes are vortexed to remove expanded cumulus cells and stained with Hoechst 33342 (2 µg/mL) for the observation of DNA (chromatin). To make sure the enucleation, ultraviolet light is used to check DNA located in the polar body and the metaphase plate. Donor cells are trypsinized, pelleted, and resuspended in DMEM supplemented with either 0.5% FCS (serum starved) or 10% FCS and 15 µM roscovitine prior to transfer into recipient oocytes. Donor cells (one per oocyte) are microsurgically placed into the perivitelline space evacuated during nucleation, ensuring intimate contact between the donor cell and the recipient oocyte (Houdebine, 2003).

Why did the chicken cross the mobius strip? To get to the same side. Nuclear transfer unit fusion and activation The donor cell and recipient cytoplasm of the nuclear transfer couplets are fused approximately 22–24 hours postmaturation by a single direct electrical pulse (40 V) delivered through needle-type electrodes. Fusion took place in Zimmermann cell fusion medium by placing an electrode on each side of the nuclear transfer couplet (approximately 0.15 mm apart) and arranging the couplet so that the 20 µsec pulse is delivered perpendicular to the shared membrane space of the donor cell/cytoplasm. A sample of couplets is examined 1 hour after the pulse to determine fusion efficiency. Activation of the couplets is performed beginning 2 hours after fusion as described previously, using TCM 199 plus 1.0% FCS supplemented with cytochalasin B (5 µg/mL), cycloheximide (10 µg/mL), and calcium ionophore (5 mM) for 10 min followed by TCM 199 plus 10.0% FCS supplemented with only cytochalasin B (5 µg/mL) and cycloheximide (10 µg/mL) for 1 hour in 5.0% CO2 and air. A 5-hours culture period in TCM 199 plus 10.0% FCS and cycloheximide (10 µg/mL) alone is conducted under low oxygen tension (5.0% CO2, 5.0% O2, 90.0% N2). Following activation, reconstructed embryos are cultured in BARC medium under low oxygen (5.0%) for 7 or 8 days (Chesne, 2002; Faurie, 2003). Don’t forget to Breathe

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Embryo transfer Embryos that reaches the blastocyst stage are transferred into recipient animals approximately 7 days after synchronized estrus. One or two embryos per recipient are nonsurgically introduced into the uterine horn ipsilateral to the ovary containing a palpable corpus luteum. Pregnancy evaluation is performed using transrectal ultrasound approximately 21 days following embryo transfer (Day 28 of gestation). Recipients diagnosed as pregnant are evaluated weekly until approximately 100 days of gestation and then monthly thereafter to study fetal development. During the last month of gestation, recipients are monitored several times each week through palpation to evaluate the health of fetus. Recipients that are ketotic in the third trimester are treated with standard protocols, including a higher protein ration and propylene glycol (De Sousa, 2002).

Calving Calving is done with a standing surgical route on approximately day 272 of gestation. Parturition induction began approximately 36 hours before scheduled cesarean section with 5 mg/kg of dexamethasone (3 i.m. injections every 12 hours) supplementedwith 25 mg of prostaglandin (i.m.) at the time of the second steroid injection. If meconium is present at the time of delivery, a more aggressive approach (either via coupage or active suction) is adopted, consisting of multiple attempts to remove fluid from the lungs. Animals are given nasal oxygen at 2–10 l/min depending upon blood gas values. Plasma (e.g., 2 l/calf, i.v.), antibiotics, and colostrum (10%–15% of body weight, either by suckling or stomach tube) are given in the first 4 hours of life. More aggressive treatments (e.g., surfactant, inhaled steroids, bronchialdilators) are administered on a per case basis. The umbilicus is surgically removed to for the infection prevention. Animals are bottle-fed and weaned at several weeks of age following standard animal husbandry practices (Kishi, 2000; Heyman, 2002).

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Cell lines The adult cell line can be derived from a surgical excisional biopsy. Thin tissue sections are cut into 1- to 3-mm pieces with a sharp surgical blade, and explants are transferred into 25-mm2 flasks containing Dulbecco’s modified Eagle’s medium (DMEM-F12) +10% fetal bovine serum (v/v) + 1% penicillin/streptomycin (v/v) (10 000 U/mL penicillin G, 10 000 µg/mL streptomycin) and then cultured at 37°C in air containing 5% CO2. When confluence is achieved at 14 days, cells are trypsinized for 5 min, and total cell count is determined using a Coulter counter. The recovered cells are centrifuged, and the pellet is resuspended at a concentration of 1 million cells per mL. Aliquots are either frozen in DMEM-F12 containing 10% dimethyl sulfoxide (DMSO) before storage at -80°C, or 250000 cells are transferred into a new 25-mm2 flask. As confluence is approached, the cells are passaged by trypsinization and again counted. The mean population doubling time for the first three passages (24 days in culture) could be 44 hours. A Day 40 fetus cloned from the adult cell line is surgically removed from the recipient cow’s uterus. The head and viscera are removed, and the remainder of the fetal tissue is sliced into 2- to 5-mm pieces. These explants are then cultured as above. The mean population doubling time for the first eight passages (44 days in culture) could be 27.4 hours (Arat, 2001;Zakhartchenko, 1999).

How do you get down off an elephant? You don’t! You get down off a duck!

Nuclear transfer Recipient oocytes could be slaughterhousederived from predominantly Brahman-cross cattle and matured for 17 hours in Medium 199 supplemented with 10% fetal calf serum, FSH (0.1 U/mL), LH (0.1U/mL), estradiol (1 µg/mL), pyruvate (28 µg/mL),EGF (0.05 µg/mL), and 1% penicillin streptomycin. The cumulus-oocyte complexes are vortexed at 17 hours postmaturation (hpm) for 1–2 min, and then the oocytes are washed, placed in 0.05% Pronase E (w/v) for 3 minutes, and held in Medium 199 + 10% FCS (Zakhartchenko, 1999; Kuhholzer, 2001).

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Enucleation Oocytes are enucleated at 19 hpm. Before enucleation, oocytes are placed for 15 minutes in Hepes-buffered synthetic oviductal fluid (H-SOF) with 4 mg/mL BSA that contained 7.5 µg/mL cytochalasin B and 5 µg/mL Hoechst 33342. At this time, oocytes are selected for presence of a polar body and homogeneous cytoplasm. Suitable ocytes are enucleated in H-SOF with 7.5 µg/mL cytochalasin B using a beveled 25-µm outside-diameter glass pipette. Only oocytes in which the removal of both the polar body and metaphase nucleus is confirmed by observation under UV light are included in the experiment. Oocytes are then randomly allocated to be combined with either adult or fetal fibroblasts (Oback, 2003). Donor cells Serum starvation of donor cells is achieved by culture in DMEM/F12 + 0.05% FCS for 1–5 days before nuclear. Fibroblasts are prepared by trypsinization of early-passage adult (passage [P] 3–4; Days 13–24 in culture) and fetal (P 3–4; Days 11–21 in culture) cells at 60–80% confluence (Clark, 2003).

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~New Splicer~ important version update. GFP will now be introduced to all new models of Dodo and Frogs (exotic and unnatural). GFP containing Lenti-Viruses and subsequent transduction to cells was today perfected and enjoyed in the dark this evening. They look pretty too, pictures to follow. New orders can be obtained by choosing luminous Green, pink, yellow and red can be selected from the drop down bar in the ordering menu. Special OFFER ~ Buy all 4 colours and not only identify all your dodos/frogs in the dark! The 5th order can be dual transfected with your favourite colour! ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

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Combining donor and recipient cells Fibroblasts of median diameter are combined with enucleated oocytes in 7.5 µg/mL cytochalasin B in HSOF using a 30-µmoutside-diameter glass pipette, then returned to Medium 199 + 10% FCS. The oocytefibroblast couplets are manually aligned and fused in a 3.2-mm fusion chamber that contained Zimmerman cell fusion medium using 2 × 20-µsec 1.6-kV/cm DC fusion pulses delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA). Oocyte activation is performed 3–5 hours after fusion at 27 hpm, by a 4-min incubation in 5 µM ionomycin followed by 4 min in 3% BSA in Tyrode’s lactateHepes and 4 min in H-SOF. Fusion is assessed at this time by light microscopy before transfer into 100 µM butyrolactone-I in SOF for 4 hours followed by transfer to Charles Rosenkrans medium #1 with added amino acids (CR1aa) + 10% FCS with buffalo rat liver coculture for 7 days (Zhu, 2002). Embryo development Embryos are classified as blastocysts according to their morphology on Day 7 or Day 8 following NT. Two or three blastocysts are nonsurgically transferred when synchronized recipients are available. Pregnancy status is assessed by transrectal ultrasonography Aloka 500, 5-MHz transducer; Aloka Co., Tokyo, Japan) at 30 days post-nuclear transfer and confirmed 10 days later, with pregnant recipients rechecked every 2 weeks (Hill, 2000). Briefly, the protocol of animal clone by nuclear transfer could be summarized in Figure 1.

Don’t forget to Breathe

Don’t forget to smile


And if all that fails..... Be a Dodo... “Raphus cucullatus doesn’t exist... Said the Dodo... Before it was too late and he perished...” ~New Splicer~ CUT HERE - - - -

CUT HERE - - - -

CUT HERE - - - -

Mr. Quaverley and Jasper Fforde will never be cloned. (not until its safe or cheaper to do so).

Don’t forget to Breathe

Don’t forget to smile


Next Volume... MiniDinoSaurs!!!

By popular demand of two people one of whom was not me!!!! MDS or myelodysplastic syndromes as we like to call them

Why does a dinosaur climb a tree? To get in his nest.

Don’t forget to Breathe

Don’t forget to smile


Thanks for listening, thanks for smiling...

Next time this could be your pet! Stay tuned to ~New Splicer~ For all your cloning needs and desires that even you didn’t know you had!

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Buy TOAST !!! Lasts that bit longer than butter!

Why does a dinosaur climb a tree? To get in his nest.

Toast Marketing board Don’t forget to Breathe

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New Splicer Volume 1.0  

Originally inspired by the fictional home-cloning plot devices in Jasper Fforde's Thursday Next series of books. Please help this page grow...

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