Genes and Even More Things 2          original text from New Scientist June 10, 2006

False dawn
1) Back in the 1980s, molecular biologists thought they had cracked it. They found that if they took mouse embryonic stem cells (ESCs) and added a new DNA sequence flanked by mouse DNA, overeager repair enzymes would sometimes cut the matching sequence out of the mouse genome and splice in the new DNA instead, a phenomenon called homologous recombination. Exactly why this happens remains a mystery, and it only works in around 1 in 100,000 cells. But it works.

2) Thanks to homologous recombination, genetic engineers now have exquisite control over the mouse genome. The procedure is so routine that the European Union has set up a project to study 20,000 mouse genes - two-thirds of the total - by creating 20,000 modified strains in just three years. No wonder mice are so popular with geneticists.

3) However, when biologists tried to make homologous recombination work in other animals, they got a nasty shock. For reasons that nobody fully understands, the phenomenon is exceedingly rare in just about every cell type other than mouse ESCs. That makes it impractical for genetic engineering and a complete non-starter for gene therapy. So while geneticists perform ever more spectacular tricks with mice, the "throw it in and hope" approach has remained the only way to modify most animals and plants.

4) Now this is changing. Genetic engineers are developing several methods for making precise changes to the DNA of living cells. These methods are also far more efficient, transforming up to 1 in 5 cells.

5) One relies on the tricks evolved by viruses that infect bacteria. Many of these can precision-engineer their hosts' DNA, integrating their genes at specific target sites with the help of enzymes called recombinases.

6) Recombinases work by recognising two distinct sequences: one on the viral DNA to be spliced into the host, and a second at the target site on the host's genome (see Diagram). The enzyme cuts the DNA at both sites and stitches the whole lot together. This ability gives recombinases enormous potential for genetic engineering. Simply add the first target sequence to any piece of circular DNA and the recombinase will integrate it into any other piece of DNA containing the second target.

7) The catch is that because recombinases evolved to target bacteria, most plant and animal genomes do not contain the second target. But geneticists have realised this need not stop them: if an organism does not have a target sequence, they can add one using old-fashioned random integration. That might sound like a return to square one, because you cannot determine where the target sequence lands, but it's not. All you have to do is create one animal with the target in a good location, and you can then breed it and add any gene to that location in the offspring. "You know exactly what's happening, where it's going," says Eggleston. "It's like a cassette deck."

8) Recombinases are becoming increasingly popular with genetic engineers. Eggleston has been working with a particularly promising one called phi C31. His team's overall goal is to engineer mosquitoes to make them unable to transmit the malaria parasite. Last year, his team created several strains of mosquito with the phi C31 target sequence. Using the recombinase, they can now add different genes to the same spot time and again, greatly improving the reliability of their results. This method could be used to modify a wide range of animals, and possibly plants too.

9) When it comes to gene therapy, however, adding a "cassette deck" in advance is not an option. Fortunately Michele Calos of Stanford University in California, who pioneered the use of phi C31, has shown that it is not necessary. She found that phi C31 can add DNA to "pseudo sites" with only a 30 per cent match to the target. The integration process is less efficient, but chance alone dictates that the genomes of many animals will include at least one pseudo site. In 2002, her team proved that phi C31 could be used for gene therapy. They used the recombinase method to add a gene for a clotting agent called Factor IX - the lack of which causes one kind of haemophilia - to the livers of mice. Soon many of the mice's liver cells were pumping out factor IX, enough to cure the disorder (Nature Biotechnology, vol 20, p 1124). "It was the first study we did and it worked first time," Calos says. "We have had a lot of successful trials in animals."

10) The challenge is proving it will be safe in people. Phi C31 inserts DNA at two main sites in the mouse genome, but in human cells it is less discriminating. Calos has identified 19 main integration sites and another 82 where it occurs from time to time. "That is wider than we would like," she admits. Even so, having 101 known integration sites is a massive improvement on random insertion. Calos also plans to "evolve" recombinases that are more specific by mutating the gene for phi C31.

11) Another cause for concern is a recent study showing that very high levels of phi C31 can trigger chromosomal rearrangements in human cells. Random rearrangements of this kind can cause cancer. Calos points out, however, that the phi C31 rearrangements are not random: they occur at the integration sites, none of which are near cancer-related genes. "We may never be able to eliminate off-target activity completely," she says. "But the bottom line is that we are not seeing any adverse events or tumours in animals."

12) Calos hopes clinical trials will begin within two years, with haemophilia likely to be the first disease targeted. There have also been successful tests in mice or human cells of treatments for a hereditary skin disorder, a liver disease called tyrosinemia and X-SCID, the "bubble boy" immune disease.

13) The ultimate goal, though, is to target any sequence, rather than merely deliver DNA to a handful of predetermined sites. Why? Because many genetic disorders are caused not just by the absence of a normal gene, but by the presence of a faulty one. In these cases, you have to fix the spelling mistake; it is not enough to add a corrected "sentence".

14) Enter biotech company Sangamo Biosciences of Richmond, California. Sangamo specialises in designing "zinc fingers" - naturally occurring substructures found within most DNA-binding proteins that lock onto specific DNA sequences. In theory, by using novel combinations of zinc fingers, you can make custom proteins that will bind to any sequence you choose.

Task 1 Vocabulary: Find the words listed in the table below in the text and decide what they mean.
Word or phrase
 Meaning in text
1 ‘cracked it’ in para 1

2 ‘phenomenon’ in para 1
3 'exquisite' in para 2
4  ‘impractical’ in para 3
5  ‘precise’ in para 4
6  ‘integrating’ in para 5
7  ‘spliced’ in para 6
8  ‘return to square one’ in para 7
9  ‘pseudo’ in para 9
10‘trigger’ in para 11

Task 2 Comprehension: answer the questions below.
Q1
 What is homologous recombination?
A
Q2 Why does it happen?
A
Q1 What is the problem with homologous recombination?
A
Q3 What are ESCs?
A
Q4 By how much are the methods scientists are developing now better than homologous
recombination?
A
Q5 What are recombinases?
A
Q6 How do they operate?
A
Q7 What is the problem with recombinases?
A
Q8 What is phi C31?
A
Q9 Why is it easier to successfully insert DNA using phi C31 into mice than it is with humans?
A
Q10 What is one possible problem of using phi C31 on humans?
A
Q11 What is another name for X-SCID?
A
Q12 What is Sangamo?
A
Q13 What are zinc fingers?
A
Q14 The subtitle of this subsection is False Dawn. Why do you think the author chose that title?
A


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