Clonación 

La clonación (del griego κλών, "retoño, rama") puede definirse como el proceso por el que se consiguen, de forma asexual, copias semejantes de un organismo, célula omolécula ya desarrollado.

Se deben tomar en cuenta las siguientes características

• En primer lugar se necesita clonar las moléculas, ya que no se puede hacer un órgano o parte del "clon" si no se cuenta con las moléculas que forman a dicho ser.
• Ser parte de un animal ya "desarrollado", porque la clonación responde a un interés por obtener copias de un determinado animal que nos interesa, y sólo cuando es adulto conocemos sus características.
• Por otro lado, se trata de crearlo de forma asexual. La reproducción sexual no nos permite obtener copias idénticas, ya que este tipo de reproducción por su misma naturaleza genera diversidad.

Clonación molecular

La clonación molecular se utiliza en una amplia variedad de experimentos biológicos y las aplicaciones prácticas van desde la toma de huellas dactilares a producción de proteínasa gran escala.

En la práctica, con el fin de amplificar cualquier secuencia en un organismo vivo, la secuencia a clonar tiene que estar vinculada a un origen de replicación; que es una secuencia de ADN.

Transfección

Se introduce la secuencia formada dentro de células.

Selección

Finalmente se seleccionan las células que han sido transfectadas con éxito con el nuevo ADN.

Inicialmente, el ADN de interés necesita ser aislado de un segmento de ADN de tamaño adecuado. Posteriormente, se da el proceso de ligación cuando el fragmento amplificado se inserta en un vector de clonación: El vector se linealiza (ya que es circular),usando enzimas de restricción y a continuación se incuban en condiciones adecuadas el fragmento de ADN de interés y el vector con la enzima ADN ligasa.

Tras la ligación del vector con el inserto de interés, se produce la transfección dentro de las células, para ello las células transfectadas son cultivadas; este proceso, es el proceso determinante, ya que es la parte en la que vemos si las células han sido transfectadas exitosamente o no.

Tendremos que identificar por tanto las células transfectadas y las no transfectadas, existen vectores de clonación modernos que incluyen marcadores de resitencia a losantibióticos con los que sólo las células que han sido transfectadas pueden crecer. Hay otros vectores de clonación que proporcionan color azul/ blanco cribado. De modo, que la investigación de las colonias es necesaria para confirmar que la clonación se ha realizado correctamente.

Clonación celular

Clonar una célula consiste en formar un grupo de ellas a partir de una sola. En el caso de organismos unicelulares como bacterias y levaduras, este proceso es muy sencillo, y sólo requiere la inoculación de los productos adecuados.

Sin embargo, en el caso de cultivos de células en organismos multicelulares, la clonación de las células es una tarea difícil, ya que estas células necesitan unas condiciones del medio muy específicas.

Una técnica útil de cultivo de tejidos utilizada para clonar distintos linajes de células es el uso de aros de clonación (cilindros).

De acuerdo con esta técnica, una agrupación de células unicelulares que han sido expuestas a un agente mutagénico o a un medicamento utilizado para propiciar la selección se ponen en una alta dilución para crear colonias aisladas; cada una proviniendo de una sola célula potencialmente y clónicamente diferenciada.

En una primera etapa de crecimiento, cuando las colonias tienen sólo unas pocas células; se sumergen aros estériles de poliestireno en grasa, y se ponen sobre una colonia individual junto con una pequeña cantidad de tripsina.

Las células que se clonan, se recolectan dentro del aro y se llevan a un nuevo contenedor para que continúe su crecimiento en forma natural.

Clonación de organismos de forma natural

La clonación de un organismo es crear un nuevo organismo con la misma información genética que una célula existente. Es un método de reproducción asexual, donde lafertilización no ocurre. En términos generales, sólo hay un progenitor involucrado. Esta forma de reproducción es muy común en organismos como las amebas y otros seres unicelulares, aunque la mayoría de las plantas y hongos también se reproducen asexualmente.

También se incluye la obtención de gemelos idénticos de manera natural. Se considera como una alteración espontánea durante el desarrollo embrionario, ignorándose su causa, aunque existe una correlación familiar estadísticamente significativa.

Consideraciones éticas a la clonación

Argumentos en favor de la clonación humana terapéutica

El aumento de la esperanza de vida de los seres humanos ha provocado un notable aumento de las enfermedades crónicas o degenerativas, como las enfermedades cardíacas, elalzheimer o el cáncer. El principal problema es que estas enfermedades afectan a partes del organismo que, debido a un aumento de la longevidad o al daño irreversible sufrido, el cuerpo no puede regenerar por sí solo. Una solución a estas enfermedades puede ser la clonación terapéutica, al ser una especialización del tratamiento con células madre. Cuando un órgano o tejido ha sido dañado es necesario regenerarlo o realizar un trasplante, pero los trasplantes tienen varias dificultades, como la dificultad para encontrar donantes, el posible rechazo inmunitario o la imposibilidad de trasplantar ciertos tejidos u órganos.

La clonación terapéutica ofrece grandes posibilidades, aún en investigación, para aplicarse en sustitución a los trasplantes u otras terapias poco efectivas contra enfermedades graves. La obtención de células embrionarias de un individuo, para utilizarlas en beneficio de su propia salud, supone una posibilidad de curación que es tomada en consideración, por el derecho a la salud que tienen los seres humanos, según la Organización Mundial de la Salud.

Cloning

loning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.

The term clone is derived from the Ancient Greek word κλών (klōn, “twig”), referring to the process whereby a new plant can be created from a twig. In horticulture, the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.

In botany, the term lusus was traditionally used.^]

In the United States, the human consumption of meat and other products from cloned animals was approved by the FDA on December 28, 2006, with no special labeling required. Cloned beef and other products have since been regularly consumed in the US without distinction. Such practice has met strong resistance in other regions, such as Europe, particularly over the labeling issue.

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations.

Cloning of any DNA fragment essentially involves four steps

1. fragmentation - breaking apart a strand of DNA
2. ligation - gluing together pieces of DNA in a desired sequence
3. transfection - inserting the newly formed pieces of DNA into cells
4. screening/selection - selecting out the cells that were successfully transfected with the new DNA

Although these steps are invariable among cloning procedures a number of alternative routes can be selected, these are summarized as a 'cloning strategy'.

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Cellular cloning

Unicellular organisms

Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

Cloning in stem cell research

Somatic cell nuclear transfer, known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning." The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.

Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer’s. The process begins by taking out the nucleus that contains the DNA from an egg and putting it in a nucleus from an adult. In the case of someone with Alzheimer’s disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient. The embryo will then form a blastocyst which has the potential to form/become any cell in the body.

The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the oocyte is inserted into the donor’s somatic cell nucleus. The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells.

SCNT Process The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use.The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm. This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current. This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals.

Applications of SCNT SCNT is seen to be a great method for producing agriculture animals for food consumption. It successfully cloned sheep, cattle, goats, and pigs. Another benefit is SCNT is seen as a solution to clone endangered species that are on the verge of going extinct.

Limitations of SCNT Stresses placed on both the egg cell and the introduced nucleus are enormous, leading to a high loss in resulting cells. For example, Dolly the sheep was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood. As the procedure currently cannot be automated, but has to be performed manually under a microscope, SCNT is very resource intensive. The biochemistry involved in reprogramming thedifferentiated somatic cell nucleus and activating the recipient egg is also far from understood.

In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus.

Human cloning

Human cloning is the creation of a genetically identical copy of an existing or previously existing human. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction. There are two commonly discussed types of human cloning: therapeutic cloning and reproductive cloning. Therapeutic cloning involves cloning adult cells for use in medicine and is an active area of research. Reproductive cloning would involve making cloned humans. A third type of cloning called replacement cloning is a theoretical possibility, and would be a combination of therapeutic and reproductive cloning. Replacement cloning would entail the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant.

The various forms of human cloning are controversial. There have been numerous demands for all progress in the human cloning field to be halted. Most scientific, governmental and religious organizations oppose reproductive cloning. The American Association for the Advancement of Science (AAAS) and other scientific organizations have made public statements suggesting that human reproductive cloning be banned until safety issues are resolved. Serious ethical concerns have been raised by the future possibility of harvesting organs from clones. Some people have considered the idea of growing organs separately from a human organism - in doing this, a new organ supply could be established without the moral implications of harvesting them from humans. Research is also being done on the idea of growing organs that are biologically acceptable to the human body inside of other organisms, such as pigs or cows, then transplanting them to humans, a form of xenotransplantation.

The first hybrid human clone was created in November 1998, by Advanced Cell Technologies. It was created from a man's leg cell, and a cow's egg whose DNA was removed. It was destroyed after 12 days. Since a normal embryo implants at 14 days, Dr Robert Lanza, ACT's director of tissue engineering, told the Daily Mail newspaper that the embryo could not be seen as a person before 14 days. While making an embryo, which may have resulted in a complete human had it been allowed to come to term, according to ACT: "[ACT's] aim was 'therapeutic cloning' not 'reproductive cloning'"

On January, 2008, Wood and Andrew French, Stemagen's chief scientific officer in California, announced that they successfully created the first 5 mature human embryos usingDNA from adult skin cells, aiming to provide a source of viable embryonic stem cells. Dr. Samuel Wood and a colleague donated skin cells, and DNA from those cells was transferred to human eggs. It is not clear if the embryos produced would have been capable of further development, but Dr. Wood stated that if that were possible, using the technology for reproductive cloning would be both unethical and illegal. The 5 cloned embryos, created in Stemagen Corporation lab, in La Jolla, were destroyed.