Trends in Biotechnology

Revision Questions

In 2005, ES-like cells were generated using four factors. The resulting cells were called iPS cells.

What does iPS mean?

induced pluripotent stem cells = iPS cells

What are some different sources of stem cells?

Embryonic stem cellsNuclear transfer cellsAdult stem cellsInduced pluripotent stem cells

Nuclear reprogramming was first proved by Sir John Gurdon in 1962.

What did Gurdon do?

Gurdon generated new frogs by transferring tadpole intestine cell nuclei into enucleated eggs from the African clawed toad, Xenopuslaevis.

1997, Sir Ian Wilmut's team showed Dolly the sheep, the first cloned mammal created using a nuclear transfer method. Why is this significant?

It showed that the genome DNA of mature cells theoretically have all the information needed to develop animals, and that this DNA can be reprogrammed.

In 2001 Takashi Tada demonstrated that thymocytes acquire pluripotency upon electrofusion with mouse ES cells. What does this show?

It showed that ES cells also contain factors that induce pluripotency in somatic cells.

Shinya Yamanaka’s lab identified many factors that either are specifically expressed by or have important roles in mouse ES cells.

What did many of these factors seem to be?

They were transcription factors.

Which four transcription factors were essential for developing pluripotent cells?

Oct3/4, Sox2, Klf4 and c-Myc

How were the genes for the transcription factors put into the target cells?

A lentiviral vector.

In 2007 Thomson's lab announced in Science that they had also succeeded in making human iPS cells using a different set of four factors - Nanog, Lin28, Oct3/4 and Sox2. Later Yamanaka showed that iPS cells can be generated without c-Myc.Why was this important?

c-Myc is an oncogene

X-ray crystallography can be used to find out the structure of molecules.If we want carry out a crystallographic study what do we need to have?

A crystallized sample of pure protein.

What causes chronic myelogenous leukemia(CML)?

A chromosomal translocation creating an abnormal fusion protein, kinase BCR-ABL, which signals wrongly, leading to uncontrolled proliferation of the leukemia cells.

What is tyrosine kinase?

Tyrosine kinase is a common signaling molecule that, when activated, triggers cells to divide. In CML, the mutated tyrosine kinase is active for too long, causing cells to multiply at an abnormally high rate. This results in the overproduction and accumulation of immature white blood cells.

Scientists found considerable variation in the ATP-binding pockets of the different kinases.What does this mean for treatment?

This meant that a drug that specifically blocks the bcr-abl ATP binding site might be possible.

How does Imatinib work?

Imatinib works by preventing a tyrosine kinase enzyme, in this case BCR-Abl, from phosphorylating subsequent proteins and initiating the signalling cascade necessary for cancer development, thus preventing the growth of cancer cells and leading to their death by apoptosis.

In the 1990s, Andrew Fire and Craig Mello were studying how gene expression is regulated in C. elegans. Among other things, they were examining mRNA that encodes a protein involved in the nematode’s ability to move. They injected the nematode gonads with sense RNA.What is sense RNA?

A molecule identical to a segment of mRNA

Fire and Mello injected other nematodes with antisense RNA that could bind to the corresponding mRNA for the muscle protein.Which RNA caused a major change in the muscles, sense or antisense?

Neither RNA had much effect.

When they injected a mixture of sense RNA and antisense RNA, the nematode offspring moved in an odd, twitching fashion. Similar movement patterns could be seen in nematodes with a defective muscle protein gene. What was the explanation for this surprising finding?

Together, sense RNA and the corresponding antisense RNA make up double-stranded RNA, and it seemed as if this could silence the gene that encoded the muscle protein.These RNA molecules control degradation of mRNA molecules by biochemical machinery.

What is RNA interference naturally used for?

Regulation of gene expression.Defence against viruses.Protection against transposons.