Birth dating of cells is important to fully elucidate when a cell has been created, to determine what factors or conditions may have led to its conception. Birth dating has been applied within many cell constructs, though recent attention has focused on cells within the central nervous system. With the relatively recent discovery of neuro-genesis, a process of creating functionally integrated neurons from progenitor cells, a plethora of innovative techniques to track the rate of cell birth have been developed.
Among some of the most widely used techniques for birth dating of cells are analyses based on the incorporation of nucleotide analogs during cell division, expression of specific markers during the maturation process, and genetic marking with retroviruses. The most robust and reliable results for birth dating are generated from nucleo-tide analogs and genetic marking by retrovirus, whereas the expression of specific markers has elicited relatively poor results. Birth dating of cells by retrovirus has stimulated a great deal of interest because of the ability to visualize tissue directly, as opposed to the nucleotide analog methodology, which requires tissue fixation and DNA denaturing. Though the retroviral method is invasive, many researchers feel that the pros outweigh the cons of this newly developed birth dating technique.
Within the paradigm of neurogenesis, it is extremely important to understand when a functional neuron has been created. This information is critical, as it allows researchers to focus on aspects of the microenvironment at a specific time point that led to the production of new cells. This essentially enables researchers to “rewind time” to reliably chart cell birth and development in a living system. As a consequence, important facts about neurogenesis can be collected, such as the region of the brain in which this process is occurring, particular factors/neurotransmitters that are present in the microenvironment at the appropriate time, genes that may be upregulated or downregulated, and so on. Birth dating of cells by retrovirus can provide all of these details in a living system, which has afforded the most convincing evidence thus far that newborn neurons in the adult mammalian central nervous system are in fact functional and physiologically active. The ability of a retrovirus to integrate into normally functioning tissue offers a tremendous advantage compared with other techniques.
To fully comprehend the use of a retrovirus to date the birth of a cell, one must understand the properties of a retrovirus. Retroviruses are enveloped viruses with an RNA genome that replicate via a DNA intermediate. Retroviruses rely on the enzyme reverse transcriptase to reverse transcribe its genome from RNA into DNA, which can then be integrated into the host’s genome using the enzyme integrase. The virus then replicates as part of the host cell’s DNA. Furthermore, there are many different subfamilies of retroviruses that each have different properties. For example, the oncovirus subfamily of retroviruses depends on host cell proliferation for completion of the viral life cycle, whereas the lentivirus subfamily replicates without this process. It is important for researchers to understand the intricacies of the retroviral vector they choose, as some vectors rely on the breakdown of the host cell’s nuclear membrane for integration, whereas some possess the appropriate nuclear import abilities so that integration into the host cell’s genome can occur at all times.
Analysis of cell birth based on genetic marking with retroviruses involves the expression of trans-genes from retroviruses. Transgenes are genes that are taken from the genome of the retrovirus and introduced into the genome of the cell of interest. For this mechanism to take place, viral integration into the host genome must occur. Some retrovi-ruses, such as the Muloney murine leukemia virus, lack nuclear import mechanisms, causing the ret-rovirus to limit viral integration to when the host nuclear membrane dissolves during mitosis. Ultimately, this allows such a retrovirus to serve as a sufficient marker of host cell division. Expression of a live reporter such as green fluorescent protein (GFP) allows direct visualization of fully functioning newborn cells. GFP fills the cell body of the neuron or cell of interest, making structural analysis an additional possibility. To appropriately label proliferating cells, a highly concentrated retroviral stock carrying the GFP transgene must be administered. Though retroviral labeling can be quite variable, there is usually a relatively high percentage of GFP cells expressed.
PROS AND CONS
Birth dating of cells through use of a retrovirus has many pros and cons. In terms of benefits, this method has elicited some of the most robust results to date concerning analysis of adult neu-rogenesis in living creatures. It is a specific procedure that facilitates identification of the few newborn neurons surrounded by billions of preexisting neurons in the adult central nervous system. Retroviral dating of recently created cells also allows direct visualization and analysis of such cells through use of the GFP live reporter. This is a major benefit, as one can observe living cells functioning in their normal environment as opposed to requiring cells to be sacrificed and subsequently fixed. Furthermore, whole-cell morphology can be analyzed using this method. Although the nucleotide analog method of birth dating is limited to the nuclear region of the cell of interest, genetic marking by retrovirus can truly evaluate all aspects of the cell. Again, GFP facilitates this process, as it is a live reporter, in addition to serving as a protein that permeates the cell body of a neuron.
Although this method does possess many benefits, there are some significant disadvantages to its use. The first and most apparent disadvantage to this method involves the invasive stereotaxic injection into specific brain regions. Considering that the majority of mammalian neurogenesis occurs in three deep-set regions of the brain, the actual administration of the retrovirus into these regions is challenging. The three regions of interest are the subventricular zone of the lateral ventricles, the subgranular zone of the dentate gyrus in the hippocampus, and the more rostral olfactory bulbs.
A needle must directly penetrate these regions so that high doses of retrovirus may be administered in a deliberate and intentional manner. As a result, although the retrovirus itself may be specific, the likelihood of experimental error during direct administration is high. Finally, birth dating of cells by retrovirus presents a risk of infection in the host organism. Any direct penetration of the skull and brain tissue of a mammal presents immunologi-cal challenges that may affect the corresponding results of the experiment.
In summary, birth dating of cells by retrovirus has become a reliable and innovative method for investigating adult neurogenesis. Although researchers are satisfied with results obtained from this single-cell genetic technique thus far, future goals include combining small inhibitory RNA—a short sequence of RNA that can be used to silence gene expression—to generate even more specific results.
