Cloning

Clones are organisms that have identical genetic material. In other words, the sequence of bases n their DNA is exactly same. Long before the birth of Dolly the sheep, clones had bee observed in both nature and in the laboratory.When a couple has an identical twin or identical triplets, the children are clones of one another.A plant cutting can also be used to generate a clone.

Prior to 199, it was thought that cloning an entire animal could only be done with embryonic cells-cells present in the early stages of an organism’s development. In the 1950's, scientists generated entire frogs from embryonic frog cells.






After a small number of cell divisions, embryonic cells start to change into the different types of cells that form muscle, blood, liver, etc. This process is called differntiation. Although each of these cells has the same genetic material, each cell can only access the genes needed for its particular function.

Before the experiment at the roslin institute, it was thought that once cells differentiated, they could not be used to generate an entire organism, for instance, in sheep udder cells could generate other udder cells, but not an entire sheep.

The scientist of roslin institute solved this problem by growing sheep udder cells under starvation conditions, this put the cells in a state similar to embryonic cells. This is called the G0 state.

An egg cell was taken from another sheep. The nucleus (which contains the genetic material) was removed from the egg cell using fine needle. They then used electric shock to fuse one starved udder cell with one nucleus free egg cell. They made 277 of these fused cells.

Although the egg cell came from a black-faced sheep, notice that the nucleus with the genetic material came from the white-faced sheep.


The fused egg cell was then inserted into several different sheep. These surrogate mothers also black-faced.

Of the 277 fused cells, only one progressed to form a developed lamb. Dolly was born on July 5, 1996.Scientist found that dolly had same DNA as the udder cells she came from. She is a clone of these udder cells.Dolly has given birth to a lamb named Bonnie, produced the natural way.Other lambs have been born at the roslin institute through their cloning process, some carry genes that will produce usable human drugs.

A laboratory in Hawaii run by Dr.Ryuzo Yanagimachi was the second group to successfully clone an animal from an adult cell. They cloned mice using cumulus cells, a cell type found in the ovaries.

The cloning method used by the lab in Hawaii was different in two ways from the method used to clone Dolly. First, the cells used to clone the mice were not grown in culture, but instead were used immediately.

Second the nucleus was removed from the cumulus cell and then directly injected into the egg cell. This egg cell's nucleus had already been removed.

The yabagimachi lab used coat color to track genetic heritage. The cumulus cell comes from an agouti (brown) mouse, and the cell comes from a black mouse.

The egg cell now had the same genetic information as the nucleus donor mouse. The egg cell was then activated and implanted into a white host mother. On October 3, 1997 the host mouse gave birth to cumulina, named after the cumulus cells she was cloned from.


Cumulina is the same color as the mouse that donated the nucleus. The DNA fingerprinting confirmed that cumulina had the same DNA as the nucleus donor.

The scientist has taken cells from cumulina to make more clones. They have successfully made several generations of clones and all mice seem normal.Dolly the sheep died at the age of 6. Since the world said hello to Dolly, Several other animals have also been cloned.

Both Dolly and cumulina were cloned from cells in the female reproductive system; cows have also been cloned using ovary and cumulus cells with the same method that was used to clone Dolly.Pigs have been added to the cloned animal menagerie. Scientist hopes to use cloned pigs to grow organs that can be transplanted into humans.













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Somatic cell nuclear transfer(SCNT)

Somatic cell nuclear transfer (SCNT) is a laboratory technique for creating an ovum with a donor nucleus . It can be used in embryonic stem cell research, or in regenerative medicine where it is sometimes referred to as "therapeutic cloning." It can also be used as the first step in the process of reproductive cloning.



Process



In SCNT the nucleus, which contains the organism's DNA, of a somatic cell (a body cell other than a sperm or egg cell) is removed and the rest of the cell discarded. At the same time, the nucleus of an egg cell is removed. The nucleus of the somatic cell is then inserted into the enucleated egg cell. After being inserted into the egg, the somatic cell nucleus is reprogrammed by the host cell. The egg, now containing the nucleus of a somatic cell, is stimulated with a shock and will begin to divide. After many mitotic divisions in culture, this single cell forms a blastocyst (an early stage embryo with about 100 cells) with almost identical DNA to the original organism.

SCNT in stem cell research

Some researchers use SCNT in stem cell research. The aim of carrying out this procedure is to obtain stem cells that are genetically matched to the donor organism. Presently, no human stem cell lines have been derived from SCNT research.


Human Embryonic Stem cell colony on mouse embryonic fibroblast feeder layer.


A potential use of genetically-customized stem cells would be to create cell lines that have genes linked to the particular disease. For example, if a person with Parkinson's disease donated his or her somatic cells, then the stem cells resulting SCNT would have genes that contribute to Parkinson's disease. In this scenario, the disease-specific stem cell lines would be studied in order to better understand the disease.


In another scenario, genetically-customized stem cell lines would be generated for cell-based therapies to transplant to the patient. The resulting cells would be genetically identical to the somatic cell donor, thus avoiding any complications from immune system rejection.


Only a handful of the labs in the world are currently using SCNT techniques in human stem cell research. In the United States, scientists at the Harvard University Stem Cell Institute, the University of California San Francisco, and possibly Advanced Cell Technology are currently researching a technique to use somatic cell nuclear transfer to produce embryonic stem cells. In the United Kingdom, the Human Fertilisation and Embryology Authority has granted permission to research groups at the Roslin Institute and the Newcastle Centre for Life. SCNT may also be occurring in China.

In 2005, a South Korean research team led by Professor Hwang Woo-suk, published claims to have derived stem cell lines via SCNT, but supported those claims with fabricated data.Recent evidence has proved that he in fact created a stem cell line from a parthenote.



SCNT in reproductive cloning


This technique is currently the basis for cloning animals (such as the famous Dolly the sheep), and in theory could be used to clone humans. However, most researchers believe that in the foreseeable future it will not be possible to use this technique to produce a human clone that will develop to term.

Essentials of Stem cell biology

Embryonic stem cells without an egg or embryo

Researchers at Whitehead Institute for Biomedical Research in Cambridge, Mass., have manipulated mouse fibroblasts and turned them into cells with such developmental elasticity that they appear identical to embryonic stem cells.





Excerpts from the video



Embryonic stem cells have potential to provide people with donor cells which can be used transplantation medicine, one of the problems of embryonic stem cells are they are derived from the embryo and it will not be compatible with immune system of the donor, so the real goal is to generate customized embryonic stem cells, A way that it thought to be accomplished was by nuclear transfer, for example If u take skin cell from a patient and introduce nucleus from the cells into a egg for whose nucleus is removed. The egg is able to reprogram the somatic cell into an embryonic state, from this people are able to isolate customized embryonic cells and those could be new for customized transplantation therapy, there will be no immune rejection.






The problem with this approach is many, it is very complex and inefficient procedure and it only so far in animals (mice only) and secondly there lot of ethical objection in using human embryo and human egg cells for therapy or research, so goal of field is to understand how the egg accomplishes reprogramming the somatic nucleus into embryonic stage once we know the reprogramming rules we could do without the egg.

What we have done in our laboratory was to use the knowledge coming from investigating and finding of molecular circulatory of embryonic stem cells and comparing it with the somatic cells and taking some key regulators or Key switches and express those into the somatic cells .In long process (few weeks) we found that these skin cells become embryonic cells,. Signatures of the reprogrammed cells these cells were indistingusble with normal embryonic stem cells, Molecular expression pattern of the genes is identical, epigenetic stage of these cells are indistinguisable from embryonic stem cells the most important is these reprogrammed cells can do anything biologically as embryonic stem cells with same developmental potency and we tested this by introducing these cells back to embryos of form prim Eric mice and even can contribute to germline, so that it can generate fibroblast after the reprogramming process being introduced we can generate mice, from all,the test we have done ,it appears that these cells have same potential for forming all lineages of the animal but also for therapy has Embryonic stem cell have.

Stem cell therapy for ALS

Besides to the use of bone marrow stem cells for treating hemotological malignancies, which is an established chemical practice today. Bone marrow cells is also under intense investigation for regenerating various organs such as heart, liver and lung .This particular study that will taking about today is " possibly of using bone marrow cells in treating a disease ALS is investigated ALS is a lethal condition associate with the degenration of Motor Neurons in the spinal cord,Cerbral cortex and brainstem.








Actual cause of ALS is unknown. Although some ALS patients have a genetic mutation. Presently there is no cure for ALS The question of the study is.” whether bone marrow cells from healthy mice can inhibit the progression of disease in mouse model of ALS?"And subsequent question is do the stem cells cross the Blood-brain barrier? also total of such status canceled/what will attract with all possible way they for Mouse model which displays a predisposition and ALS like disease. The Mutated enzyme of human SOD1 ALS like carrying the GLY93 to alanine mutation is expressed in mouse, so that it mouse express pathology of Human ALS.Bonemarrow cells from the healthy mice are transferred to the mice with are predisposed to ALS.so the progress of ALS can be inhibited.