Insight Into Cloning

Often when uttered ‘cloning’, the first word that might come up in your mind is Dolly ‘The Sheep’, But is cloning only a process used to produce living organisms like cattle, dogs, fish and mouse? No. Cloning is a combination of processes used to genetically produce a biological system that may be as simple as a cell and tissue or complex like an entire living being.

Types of cloning

There are three types of cloning –

  • Gene cloning – Gene cloning often called molecular cloning where a target gene is inserted into a circular plasmid or vector. The plasmid is then introduced into a living organism like bacteria. The bacteria which would have taken up the DNA is selected using antibiotics, dyes or selective media depending upon the gene inserted. There are 3 basic steps involved in gene cloning:
    • Cutting the DNA at specific sites using restriction enzymes and joining the sticky ends of the cut DNA (gene of interest).
    • Selection and transformation of bacteria – Not all plasmids, BAC’s, YAC’s or expression vectors are considered as suitable. The characteristics of a suitable vector are – i) Must be small in size to allow insertion of large DNA fragments | ii) Must be self-replicating inside the host cell must contain ori regions and restriction sites for restriction endonuclease enzymes along with marker genes. A plasmid can be introduced into a bacterium using electroporation, heat shock and agitation with glass beads. This method is called transformation. A plasmid usually contains antibiotic resistance genes which help in the selection of transformed bacteria. As only the transformed cells with the plasmid will grow in a selective media containing antibiotic.
  • Production of an intended product like proteins – Once the transformed bacterial colony has been identified, we can culture them in large batches. Upon giving a suitable chemical signal (Like presence or absence of inducers) the target protein can be produced.
  • Reproductive cloning – The best example of successful reproductive cloning is Dolly. In this, scientists remove a mature somatic cell from the animal they want to clone and transfer its DNA into an enucleated egg cell – oocyte whose DNA material has been removed. The egg then develops into an early-stage embryo which can be implanted into the uterus of a foster mother. The foster mother then gives birth to a clone that is identical to the original organism. This process is called Somatic Cell Nuclear Transfer (SCNT).
  • Therapeutic cloning – Therapeutic cloning is intended to produce embryonic stem cells containing the same DNA as the donor cell. In short, it is used to create cloned embryos for experimental purposes. The cloned embryos can also be used as models for diseases and new drug testing. They can also be useful for making synthetic skin, cartilage and other tissue for burn, acid and accident victims.

Cloning and medicine

Cloning in the pharmaceutical industry has been used widely in the pre-clinical area. It is useful in drug discovery by identifying disease-related genes, their databases and genetic profiles. This leads to the identification of drug target and the usage of animals as a model for testing drugs.

Cloning, nuclear transfer techniques and transgenesis can be used to create knock-out animals that can be used to study difficult and complex diseases such as Parkinson disease and Alzheimer’s. Here therapeutic cloning can be used. The cloned embryos can be at the blastocyst stage where the embryonic stem cells are isolated. The donor cell should be originated from a diabetic or Alzheimer suffering patient. These embryonic stem cells can be used by researchers to understand the mechanics of the disease.

In certain cases, like end-stage organ failure, embryonic stem cells can be used for organ or tissue replacement. These stem cells have the ability to self-renewal and differentiation into many specialized types of cells. ESC can differentiate into three germ layers – ectoderm-responsible for the formation of skin and neurons, mesoderm-responsible for the formation of blood, cardiac cells and muscle and endoderm-responsible for the formation of the pancreas.- can help save eyesight,  Despite so many advantages, harvesting Human ESC raises several ethical and moral concerns as it involves the destruction of embryos. Hence it is illegal to date.

Production of valuable proteins and pharmaceuticals in milk (Pharming)- The use of transgenic animals for the above work has increased exponentially. The gene of interest, promoters and important transcriptional elements can be introduced into the somatic cells of the donor organism. These cells can then be fused with the enucleated egg cell. Once it reaches the blastocyst stage it is implanted into the uterus of the foster mother and the offspring grows naturally. This offspring would contain the desired characteristic like tissue-specific production of the protein desired. -antibodies, enzymes, hormones. For example – Several European Researchers from different countries created a small herd of transgenic cattle that was able to produce a huge number of special antibodies which could cure cancer.