With permission from Aliquotes v.iv (rogerb@microsoft.com)
New Scotland Bovine Labs

COW CLONING KIT

Transgenic protein purification system

Instruction Manual

Introduction to Cow System(TM):

The Cow cloning system is an advanced protocol for the purification of recombinant proteins from lactating bovine species. The system is more reliable than current sheep cloning methods, requires less starting material and is less expensive.

1) Description:

The Cow System(TM) is the most powerful system for the cloning and expression of recombinant protein. Target genes are cloned in cow plasmids under control of the bovine serum albumin promotor. The desired product can comprise more than 10% (50kg) of the animal after a few hours of fermentation, in inclusion bodies.

2) Host for cloning:

Suitable cows for cloning include the Cowalbi, which are albino, and allows the black/white screening for the library when plated on the suitable field spread with X-Grass(TM)selective medium. Note that the Scottish Highland Cows (SHC) are more sensitive to the heat shock transformation.

3) Preparation of the medium:

You need a very, very big flask, a very, very big shaker and a clothes peg for your nose. The growth medium, "Cow Dip" or C.D. broth, is made from @m grass, 0.5M hay supplement, 0.2mM ice cream (vanilla flavour), pH7.2. An oxygen carrier, such as myoglobin, can be added to increase growth and prevent drowning. Alternatively, Farmer Seah(R) provide bovine aqualungs for growth in liquid media. Always autoclave the medium in order to kill scrapie prions.

4) Transformation:

You need alt least 12kg of plamid, a vet and a big swimming pool at 37C and a sauna at 42C. It is possible to use the Farmer Kit(TM) from Farmer Seah(R). Super competent Cowalbi calves are provided with the kit. Recently, a protocol derived from Maniatis has been described by Jersey et al. (1996) using Brute Force(TM)on mature bovine.
  1. Place the cows in a room on ice and mix gently to assure that the cows are in good shape.
  2. Add 2 kg of plasmid directly in each cow using a clean glove. Rotate gently to mix.
  3. Place cows on ice for 30 minutes.
  4. Heat shock the cows by placing them in the sauna at 42C for exactly 40 seconds. Do not vortex; the cows will break the mixer.
  5. Place the cows in a cold room for 2 minutes.
  6. Add two million liters of GG medium.
  7. Incubate the cows at 37C with shaking in Earthquake zone for one hour and spread the cows on a green field(*). Note that premade libraries are available from Cowtech (Cambridge).
  8. Place cows on ice for 30 minutes.

5) Expression:

  1. To start the culture, add two tonnes of transformed cows (0.25 vol. of total bovine prep (TBP)) in the liquid culture (figure 1)
  2. Incubate with shaking the culture at 37C until OD_600 of the supernatant reaches 0.8.
  3. The cows could be separated by decantation using a shepherd and his dog, by gentle centrifugation (5g) of filtration (filter, 1m).
  4. Lyse by osmotic shock in 2M Chocolate Syrup(**) or with three medium shrapnel devices. Atomic weapons are not advised. Note that English cows are very resistant to lysis. It is possible to use a French Press.
  5. Purify the product using normal procedures. For enhanced purification,use the Cow-Lyse Kit(TM).

6) Cloned gene expression in Cow System(TM)

The plasmid included with this kit is the new pBSE, the Bovine System Expression plasmid. Other Cow-based expression systems can be used but may not over-express to the same extent.
When cloning chimaeric gene constructs, remember to use Cowalbi which are missing the gene coding for the restriction enzyme BDNA I. Cowalbi are also immunosuppressed to aid expression of hideously deforming genes.
If gene products are poor, try the Rhino Kit(TM). This incorporates a grey/white selection procedure to replace the Blue Whale Kit(TM), which has been discontinued due to poor yields and international pressures.(*) Single cloned cows could be stored as frozen at -80C or in a field in Greenland.
(**) Note that the use of chocolate syrup osmotic shock can lead to milkshake production in certain bovine species.
(***) pMooscript can be used instead for lower expression levels.
Figure 1:Fermentation conditions (Cows floating in a BIG erlenmeyer)

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