Molecular Biology Techniques – 12/02

Nuclear Transfer (a general overview)

First explored by Hans Spemann in the 1920's to conduct genetics research, nuclear transfer is the technique currently used in the cloning of adult animals. A technique known as twinning exists, but can only be used before an organism’s cells differentiate. All cloning experiments of adult mammals have used a variation of nuclear transfer.

Nuclear transfer requires two cells, a donor cell and an oocyte, or egg cell. Research has proven that the egg cell works best if it is unfertilized, because it is more likely to accept the donor nucleus as its own. The egg cell must be enucleated. This eliminates the majority of its genetic information. The donor cell is then forced into the Gap Zero, or G0 cell stage, a dormant phase, in different ways depending on the technique. This dormant phase causes the cell to shut down but not die. In this state, the nucleus is ready to be accepted by the egg cell. The donor cell’s nucleus is then placed inside the egg cell, either through cell fusion or transplantion. The egg cell is then prompted to begin forming an embryo. When this happens, the embryo is then transplanted into a surrogate mother. If all is done correctly, occasionally a perfect replica of the donor animal will be born.

In July of 1998, a team of scientists at the University of Hawaii announced that they had produced three generations of genetically identical cloned mice. The technique is accredited to Teruhiko Wakayama and Ryuzo Yanagimachi of the University of Hawaii. Mice had long been held to be one of the most difficult mammals to clone due to the fact that almost immediately after a mouse egg is fertilized, it begins dividing. Sheep were used in the Roslin technique because their eggs wait several hours before dividing, possibly giving the egg time to reprogram its new nucleus. Even without this luxury, Wakayama and Yanagimachi were able to clone with a much higher success rate (three clones out of every one-hundred attempts) than Ian Wilmut (one in 277).

Wakayama approached the problem of synchronizing cell cycles differently than Wilmut. Wilmut used udder cells, which had to be forced into the G0 stage. Wakayama initially used three types of cells, Sertoli cells, brain cells, and cumulus cells. Sertoli and brain cells both remain in the G0 state naturally and cumulus cells are almost always in either the G0 or G1 state.

Unfertilized mouse egg cells were used as the recipients of the donor nuclei. After being enucleated, the egg cells had donor nuclei inserted into them. The donor nuclei were taken from cells within minutes of the each cell’s extraction from a mouse. Unlike the process used to create Dolly, no in vitro, or outside of an animal, culturing was done on the cells. After one hour, the cells had accepted the new nucleus. After an additional five hours, the egg cell was then placed in a chemical culture to jumpstart the cell’s growth, just as fertilization does in nature.

In the culture was a substance (cytochalasin B) which stopped the formation of a polar body, a second cell which normally forms before fertilization. The polar body would take half of the genes of the cell, preparing the other cell to receive genes from sperm. After being jumpstarted, the cells develop into embryos. These embryos can then be transplanted into surrogate mothers and carried to term. The most successful of the cells for the process were cumulus cells, so research was concentrated on cells of that type.

After proving that the technique was viable, Wakayama also made clones of clones and allowed the original clones to give birth normally to prove that they had full reproductive functions. At the time he released his results, Wakayama had created fifty clones. This new technique allows for further research into exactly how an egg reprograms a nucleus, since the cell functions and genomes of mice are some of the best understood. Mice also reproduce within months, much more rapidly than sheep. This aids in researching long term results.

How to Clone a Human

Materials

· Human Tissue: Pure human cells of one tissue type, from the individual who will be cloned.

· Human Tissue Culture Media: Media in which these human cells will grow and divide.

· Minimal Human Tissue Culture Media: Media in which cells will stop dividing, and enter a state of "quiescence" without dying.

· Laboratory supplies: Incubator, Sterile Hood, petri dishes, microscopes, and tools capable of removing and implanting cellular organelles, such as the nucleus, from one cell to another.

· Unfertilized human egg cells.

· Human Egg Cell growth media: Media where fertilized eggs will grow and divide.

Procedures

1. Grow the human cells to be cloned until you have a good supply.
2. Transfer the cells to minimal media. This should allow the cells to live, but they should stop dividing and enter quiescence. This is likely the step in which the cells lose their differentiation, and revert to a more totipotent state.
3. When the cultured cells are in the quiescent state, get an unfertilized human egg cell. Remove the nucleus from this egg cell. Try to minimize damage done to this cell and discard the nucleus.
4. Take one of the quiescent cells in it's entirely, and implant it inside the coat around the egg (known as the zona pellucida) next to the egg itself.
5. Electroshock the egg. The electroshock induces the fusion of the two cells, so you should be able to tell when you've electroshocked enough just by looking at the cells. The rebooting of the human genetic program is believed to be initiated by the replacement of donor cell protien signals by egg cell protien signals, but the electroshock might assist in moving those protien signals across the nuclear membrane as well. Electroporation is a common technique for moving DNA molecules through a cellular membrane.
6. Repeat the last three steps as necessary until you have enough clones. Expect a lot of them not to survive because of cellular damage and other mishaps. Allow the embryos to grow and divide a few times in Human Egg Cell growth media.
7. Implant the embryos in human mothers where they will can be carried to term, and born normally

Polymerase Chain Reaction - Xeroxing DNA

A technique called the polymerase chain reaction (PCR) allows the DNA from a selected region of any genome to be amplified more than a million-fold, provided that at least part of its nucleotide sequence is already known. The PCR method is extremely sensitive; it can be used to detect a single DNA molecule in a sample. Trace amounts of RNA can be analyzed in a similar way by converting them to DNA sequences with reverse transcriptase.

Although DNA from various individuals is more alike than different, many regions of human chromosomes exhibit a great deal of diversity. Such variable sequences are termed "polymorphic" (meaning many forms) and provide the basis for the diagnosis of genetic disease, forensic identification, and paternity testing. The PCR technique is rapidly replacing Southern blotting for prenatal diagnosis of genetic diseases and for the detection of low levels of viral infection. It also has great promise for forensic medicine, as it allows the unambiguous identification of the human source of a single cell.

Portions of the sequence that surround the region to be amplified are used to design two synthetic DNA oligonucleotides, one complementary to each strand of the DNA double helix. These oligonucleotides bracket the region to be amplified, and serve as primers for in vitro DNA synthesis, which is catalyzed by a DNA polymerase. One primer is complementary to a sequence at the beginning of the target region, and the second is complementary to a sequence of the end of the target region on the antiparallel DNA strand.

The principle of the PCR technique is illustrated above. Each cycle of the reaction requires a brief heat treatment to separate the two strands of the DNA double helix. A subsequent cooling of the DNA in the presence of a large excess of the two DNA oligonucleotide primers allows the primers to specifically hybridize to complementary DNA sequences. The annealed mixture is then incubated with DNA polymerase and the four deoxyribonucleotide triphosphates so that the regions of the DNA downstream from the primers are selectively synthesized. For effective DNA amplification, 20 to 30 cycles of reaction are required. Each cycle doubles the amount of DNA synthesized in the previous cycle. A single cycle requires only 5 minutes, and an automated procedure permits a DNA fragment to be "cloned" in a few hours, compared to the several days required for standard cloning procedures. Initially, PCR was a tedious task that involved transferring reactions between several water baths and adding fresh polymerase for each cycle. The denaturing step also denatured the polymerase. Two important innovations were responsible for automating PCR. First, a heat-stable DNA polymerase was isolated from the bacterium Thermus aquaticus, which inhabits hot springs. Taq polymerase remains active despite repeated heating during many cycles of amplification, and thus, it needs to be added only once at the beginning of the PCR reaction. Second, automated DNA thermal cyclers were invented that control the repetitive temperature changes required for PCR.

What is gel electrophoresis?

Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties.

A gel is a colloid in a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means "to carry across." Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, exist in solution as electically charged species either as cations (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

How does this technique work?

Gel electrophoresis is a technique used for the separation of nucleic acids and proteins. Separation of large (macro) molecules depends upon two forces: charge and mass. When a biological sample, such as proteins or DNA, is mixed in a buffer solution and applied to a gel, these two forces act together. The electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules. The frictional force of the gel material acts as a "molecular sieve," separating the molecules by size. During electrophoresis, macromolecules are forced to move through the pores when the electrical current is applied. Their rate of migration through the electric field depends on the strength of the field, size and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other.

Mapping a Chromosome

1) Genetic Linkage Maps

First, researchers study the way genetic markers are inherited through families. The more often markers are inherited together, the closer together they must lie on a chromosome. Markers that are not inherited together very often must lie farther apart. Researchers use this information to make a genetic linkage map that shows the distance between markers and their order on the chromosome, as you can do on the next page.

However, a genetic linkage map doesn't necessarily tell you which chromosome the markers are on. It is like knowing that Baltimore lies closer to Washington, D.C. than to New York City, without knowing which country these cities are in. Sometimes scientists can track a marker to a chromosome because they know it is linked to a marker known to be on that chromosome. Otherwise, they can create a probe (see page 9) for a marker's DNA sequence. They add the probe to a sample of prepared chromosomes. The probe will show the location of the sequence, revealing not only which chromosome is home to the marker, but also the general location on the chromosome.

2) Physical Maps with Markers

To learn more about the chromosome itself, scientists work on physical maps. Scientists cannot study a whole chromosome at one time because it is too long and too full of information (150 million bases). They can analyze only small fragments (1,000 to 30,000 bases) at a time. Therefore, scientists cut the chromosome into tiny pieces for study. But when they do that, they lose the order of the pieces. Finding that original order is like working on a jigsaw puzzle with each piece the same color and no edge pieces! To solve this problem, scientists use many copies of the same chromosome and cut each copy in different places. They find recognizable DNA markers on each piece and then look for places where the fragments share some of the same markers. Using these overlapping sections, computers then figure out the original order of the chromosome, as you can do at the right.

3) Physical DNA Sequence Maps

The final steps in physical mapping are examining the actual DNA sequence for each fragment and then compiling the sequence of the entire chromosome. Developing computers and programs that can perform this task is one of the great challenges of the Human Genome Project and has led to a whole new field called "Informatics."

Using Maps to Find Disease Genes

Scientists use maps to find disease genes hidden among three billion bases. Genetic linkage maps tell scientists which area of the chromosome to examine. Physical maps help find the disease gene itself. Then scientists can prepare probes for genetic testing, and they can study the protein the gene makes. This knowledge may lead to better ways of treating the disease. But before you learn about these efforts, you can read about new technologies that make mapping possible.

DNA Blotting

The porous and thin nature of a gel is ideal for separating DNA fragments using electrophoresis, but as we mentioned earlier, these gels are delicate and rarely usable for other techniques. For this reason, DNA that has been separated by electrophoresis is transferred from a gel to an easy to handle inert membrane, a process called blotting. The term "blotting" describes the overlaying of the membrane on the gel and the application of a pad to ensure even contact--without disturbing the positions of the DNA fragments. In the first step, the DNA trapped in the gel is denatured--the double-stranded DNA is broken into single strands by soaking the gel in an alkaline solution. This readies the DNA for hybridization with a probe--a piece of DNA that is complementary to the sequence under investigation. A membrane, usually made of a compound called nitrocellulose, is then placed on top of the gel and compressed with a heavy weight. The DNA is transferred from the gel to the membrane by simple capillary action. This procedure reproduces the exact pattern of DNA captured in the gel on the membrane. The membrane can then probed with a DNA marker to verify the presence of a target sequence.

Autoradiography: Probing DNA

To locate a specific DNA sequence, scientists rely on the base-pairing, or "hybridization," of a short piece of DNA that is complementary to the sequence of interest. This short, single stranded piece of DNA is called a "probe" and can be tagged with either mildly radioactive nucleotides or nucleotides that are linked to a substance that emits light when exposed to certain chemicals.

If we refer back to the blotting procedure described earlier, we mentioned that after the target DNA becomes trapped in the nylon membrane, the membrane is incubated in a solution that contains a probe. In this case, the probe would be radioactively labelled. Wherever the probe sequence complements a sequence on the membrane, it will anneal, or join together, to form a region of double-stranded of DNA. The membrane is then washed to remove all unbound probe and then exposed to a piece of x-ray film. The detection of radioactively labeled molecules by exposure to an X-ray sensitive photographic film is referred to as "autoradiography". Wherever the radioactively labeled probe has annealed to the test DNA, a black spot will be appear on the film.

This method is useful for a variety of applications. For example, suppose you know the DNA sequence of a particular gene (allele) that causes a disease. Now you want to know if a certain individual carries that allele. You can do this by following the steps outline above. First, isolate some of their DNA. Separate it out on a gel. Then, perform a Southern blot followed by autoradiography. If a black spot appears on the film, it indicates the presence of the disease-causing allele in that individual.

Somatic Cell Hybridization

The term "somatic" cell refers to all the cells in an organism that have differentiated into a specific cell type; excluding germ cells, stem cells, and gametes. Somatic cell hybridization is the technique of combining two cells from different tissues or species in a cell culture, typically human and rodent, with the intent of deriving various cell lines each with a different combination of chromosomes. The hybridized cells fuse and coalesce, but their nuclei generally remain separate. However, during cell division, a single spindle is formed so that each daughter cell has a single nucleus containing sets of chromosomes from each parental line. As hybrid cells grow and divide, they tend to randomly lose many of their chromosomes until they reach a stable point. From there on out, the cell will maintain the same number and species of chromosomes in subsequent divisions. Little is known about the mechanisms behind this process, but for some reason, hybrids between humans and rodents typically shed most of the human chromosomes until only 8 to 12 of the original 46 human chromosomes remain. Yet somehow, these cells can still survive. Through the careful isolation and culture of different hybrid cell lines, researchers can create a whole set of somatic cell hybrids which, together, contain the entire complement of human chromosomes. Researchers can then use these cell lines to screen for the presence or absence of a gene or gene product (protein). For example, a researcher may test the cells ability to metabolize a particular substance or study traits of antibiotic resistance. If a cell line demonstrates an effect, the researcher can then study the chromosomes present in that particular cell line to identify the gene that confers the desired effect.