Gene Therapy
Sanger Sequencing
The DNA in question will be decomposed into pieces of random length with the ends capped with radiolabeled ddNTPs. Therefore, the final nucleotide is known for all these pieces. The strands can then be assembled by length (or weight) by using a gel, which allows the order of nucleotides to be found.
Gels are read bottom up. Remember the actual sequence is the complement of it.
Frankly, this is an outdated method and has limited practical utility. Its a reflection on the curriculum that we're still taught it. Next generation sequencing is massively parallel and much more precise.
Therapies
Somatic cell therapy
Non-reproductive cells are altered to correct a gene deficit. These corrections are not passed to offspring.
Retroviral vectors help make this therapy more efficient, but may produce harmful/immunogenic products. In addition, integration into the genome is random which could break existing genes
Derivation
Adult stem cells (ASCs) are considered multipotent and will form a few cells but not many. Multipotent cells will usually come from a true stem cell, but lose ability as it differentiates (see Hematopoeisis > Blood cell production for examples). They are difficult to identify and locate, but some antibodies will bind to them. If obtained, cloning can be achieved using Somatic Cell Nuclear Transfer (SCNT).
Induced pluripotent stems cells (iPSCs) are adult stem cells which have been converted from multipotent to pluripotent. They are created by using viral vectors to transfect genes into fibroblasts.
Important gene vectors
- c-Myc (or LIN-28)
- Klf4
- Sox2
- Oct4
Problems with this method
- Retrovirus or adenovirus required
- Genes may be oncogenic
- Genes may not be controlled (e.g. may form teratomas)
- Inserted genes may replace working tumor suppressor genes
Uses of Adult Stem Cells
- Source from healthy donor, such as with bone marrow transplants.
- Source from same (sick) patient. Will have to be dedifferentiated, modified to form the target cell type, and then reinserted.
- Source from another somatic cell (transdifferentiating) and differentiate it directly into the target cell type. For example, taking adipose tissue and turning it directly into pancreatic islets.
These can be used to treat diseases from a single gene malfunction or damage to a single cell type.