Lab equipment. © INRA, William Beaucardet

Green biotechnologies: paving new paths for agriculture

Transgenesis and homologous recombination

Recent findings on the plasticity of genomes are challenging our current understanding of genomics, and indeed the relevance of the very notion of species. The genomes of living things are constantly undergoing change: mutations occur regularly, but so does the exchange of genetic material within a cell, between nuclei, chloroplasts and mitochondria, not to mention the presence of transposable elements within the gene, called transposons or “jumping genes”. The purpose of transgenesis is to transfer a gene from one organism to another in a controlled manner. The method is used frequently in labs to verify the presumed function of a gene.

Manipulateur en biologie moléculaire. © inra, William Beaucardet
© inra, William Beaucardet

 Transgenesis revisited

New methodologies described in several recent reports on targeted insertion of transgenes are calling into question current transgenesis methods. Thanks to these new methodologies, scientists are able to modify the specific sequence of a gene without modifying its genomic environment.

Most transgenesis techniques used until now led to undirected integration of the transgene in the genome. This has had several drawbacks:

-          Multiple insertions

-          Changes in the transgene

-          Insertion in genes, disturbances of the genome

-          Position: insertion in the “silent chromatin”, thus inhibiting the transgene

-          Unstable transgenes, especially in cases of multiple insertions. Instability is the result of the risk of link formations between homologous sequences and elimination by digestion-ligation.

 The new methodologies involve the use of enzymes that are capable of cutting DNA at targeted places with great precision (meganucleases), thereby facilitating the insertion of a transgene by homologous recombination. The improvement in homologous recombination, thanks to these enzymes, is hitherto unparalleled: the success rate is 1,000 to 10,000 times better than that of previous experiments (targeted insertion can reach several percent of cells, compared with 1/100,000 or 1/1000,000 without enzymes).

Applications

-          Targeted insertion of a gene

-          Deactivation of a gene (knock out): cutting a gene and selecting a natural occurrence of defective repair. This technique is the equivalent of a targeted mutation. It is the only application that does not require the introduction of a DNA template in the nucleus

-          Or, on the contrary, repair of a gene: cutting a mutant gene and repairing it by restoring the original sequence thanks to the introduction of a normal gene as a template Eg: restoring a mutant gene that causes xerodermia (sensitivity to light) in a cell culture and grafting the restored, healthy cells in humans

-          Modification of an allele: cutting and restoring a gene by using a desired allele as a template. The result is the same as that obtained with crossbreeding but the time it takes is much shorter

-          Elimination of a fragment of DNA: cut and repair using a template without the fragment that is deemed undesirable.

Other methods of obtaining a desired gene

- Tilling begins with undirected mutations by means of chemical mutagenesis followed by the repair of mutations which affect the gene of interest. Other mutations are eliminated by back-crossing. This technique is used in model plants

The principle of tilling. © INRA
© INRA

- RNA interference significantly reduces protein content: the introduction of a small double-stranded RNA molecule triggers the deactivation of a messenger RNA molecule.

References on meganucleases for plants:

- Shukla V. K. et al. 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437-441 (29 April 2009), doi.10.1038/nature07992
- Townsend J.A. et al. 2009. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442-445 (29 April 2009) doi:10.1038/nature07845 Letter