Cloning

Cloning has 2 meanings in biotechnology. The first is cloning of animals like Dolly the sheep. The second is the type that moleculear biologists think of when they clone genes and the like using recombinant DNA technology techniques. Lets start with this first meaning.

Recombinant DNA Technology

Cloning vectors have been designed to select or screen for recombinants. Commonly used vectors are pUC or M13mp series which contain the ג  complementation region of the E. coli lacZ gene and produce blue plaques or colonies on indicator plates. Insertion of your DNA fragment into this lacZ region causes a white colony or plaque which is easily identified among a background of thousands of blue transformants.

When cloning DNA, keep the following points in mind.

• Intramolecular joining of the vector molecule to the other end of itself.  This factor can be eliminated by using molecules with heterologous ends (i.e., cleaving the vector with two restriction endonucleases that produce incompatible ends). If the molecule must be cleaved with a single endonuclease, intromoleculear joining can be prevented by removing both 5' phosphates of each molecule with calf intestinal phophatase (CIP).

• Intramolecular ligation of insert fragments does not generally affect the background of unwanted colonies. But it does influence the frequency of obtaining the correct colonies because self-ligated insert DNA can not be joined to the vector. Thus where insert DNA fragments can cyclize (particularly true with short DNA fragments), relatively high concentrations of DNA (20 to 100 ug/ml) should be used to favor intermoleculear ligation events.

• Cloning of fragments with homologous and cohesive ends (i.e., a fragment that is cut with 1 enzyme like EcoRI) is not as good as directional cloning using fragments with heterologous ends (i.e., the framgent has been cut with 2 enzymes and is joined to the vector also cut with those 2 enzymes). This will reduce self-ligation.

• It is preferably to use higher molar concentrations of insert fragments to favor the desired V-I products.

• To joining incompatible termini, one must convert such termini to blunt ends. For 5' overhangs, this is accomplished by "filling in" the end on the 3' end with the Klenow fragment of E. coli DNA polymerase I in the presence of all 4 dNTPs. For 3' overhangs, the 3' to 5' exoncuclease activity of T4 DNA plymerase or Klenow enzyme is used to remove the protruding nucleotides. The T4 enzyme is preferred because it has a much more active exonuclease but it is more expensive then the Klenow enzyme. Once the relevant ends have been blunted, T4 DNA ligase can be used to join the fragments with blunt ends. But ligation efficiency is greatly reduced in comparison to fragments with cohesive ends so higher concentrations of DNA and about 10 times more ligase is advisable. Synthetic oligonucleotide linkers facilitate the cloning of blunt ended DNA fragments and are valuable for introducing new restriction sites at dired positions. Linkers are usually obtained from commercial suppliers in the nonphosphorylated form and then phosphrylated using ATP and T4 polynucleotide kinase and then added to the ligation reaction.

• Use appropriate controls to determine the success of your experiment. So set up and analyze parallel ligation reactions, each of which lacks a single DNA component. Keep the volume of the ligation reactions constant by adding an equal volume of water for the missing DNA. Also use competent cells incubated in the absence of DNA as a control to indicate any contamination in cells of transformation buffers.

Cloning of Animals

Step 1:  Tools of the trade  Specialized glass needles are created. The needles are a thin glass tube shaped like a needle which are mounted onto a robotic arm attached to a microscope.

Step 2: Isolation of eggs and skin cells Using the microscope, a collection of cow eggs are harvested 2 days before from the ovaries of cows.

Skin cells are taken from a 6 mm wide biopsy from the ear of a cow two weeks in advance. These will be the future animal. Each cell holds a complete set of chromosomes -- all the genetic information necessary to make a new cow.

Step: 3: Removal of nucleus from egg Using the glass needle, the nucleus of each egg is removed without damaging the egg. A specialized dye with ultraviolet light helps to identify the nucleus. The envelope, called the zona pellucida, is pierced with the needle up to the point of the cell membrane. Then, gentle suction is applied to the side of egg. The needles acts like a vacuum pressed against a balloon filled with Jell-O. By controlling the suction, the nucleus flows into the tip of the needle. The cell membrane is never pierced since that would kill the egg itself.

Step 4: Fushion of egg and skin cell Using a narrow needle, one skin cell is drawn into the tip of the needle and then squirted out so that it is wedged tightly between the zona pellucida of the egg and the egg's cell membrane. Several eggs are prepared in the same manner.

The eggs are transferred to another petri dish which have 2 long electrodes running horizontally acorss a plastic platform. Using a glass needle which has its tip sealed shut, the eggs are lined up on the platform between the electrodes. There is a fluid in the petri dish which is sugar based and protects the eggs from electrical damage.

Engouh electricity is given to cause the egg and the skin cell to fuse together, thereby delivering the skin cell's nucleus into the egg. (microsecond of 100 to 200 volts)

Step 5: Ebryonic development The activated eggs are placed into an incubator. By the next morning, it will be a 2 celled embryo. Evenutally the embryos will be implanted in the uteri of surrgate cow mothers and after nine months, cloned cattle will be born.

 For more information on cloning, see U.S. Patent Nos. 6,147,276, 2,252,133 and 6,525,243

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