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A knockout moss is a kind of genetically modified moss. One or more of the moss's specific genes are deleted or inactivated ("knocked out"), for example by gene targeting or other methods. After the deletion of a gene, the knockout moss has lost the trait encoded by this gene. Thus, the function of this gene can be inferred. This scientific approach is called reverse genetics because the scientist wants to understand the function of a specific gene. In classical genetics, the scientist starts with a phenotype of interest and searches for the gene that causes this phenotype. Knockout mosses are relevant for basic research in biology as well as in biotechnology.

Scientific background

The targeted deletion or alteration of genes relies on the integration of a DNA strand at a specific and predictable position into the genome of the host cell. This DNA strand must be engineered in such a way that both ends are identical to this specific gene locus. This is a prerequisite for being efficiently integrated via homologous recombination (HR). This is similar to the process used for creating knockout mice. So far, this method of gene targeting in land plants has been carried out in the mosses Physcomitrella patens and Ceratodon purpureus,^[2] since in these non-seed plant species the efficiency of HR is several orders of magnitude higher than in seed plants.^[3]

Knockout mosses are stored at and distributed by a specialized biobank, the International Moss Stock Center.

Method

For altering moss genes in a targeted way, the DNA-construct needs to be incubated together with moss protoplasts and with polyethylene glycol (PEG). Because mosses are haploid organisms, the regenerating moss filaments (protonemata) can be directly assayed for gene targeting within six weeks when utilizing PCR methods.^[4]

Examples

Chloroplast division

The first scientific publication in which knockout moss was used to identify the function of a hitherto-unknown gene appeared in 1998, and was authored by Ralf Reski and coworkers. They deleted the ftsZ-gene and thus functionally identified the first gene pivotal for the division of an organelle in any eukaryote.^[5]

Protein modifications

Physcomitrella plants were engineered with multiple knockouts to prevent the plant-specific glycosylation of proteins, an important post-translational modification. These knockout mosses are used to produce complex biopharmaceuticals in the field of molecular farming.^[6]

Mutant collection

In cooperation with the chemical company BASF, Ralf Reski and coworkers established a collection of knockout mosses to use for gene identification.^[1]^[7]

References

^ ^a ^b Egener, Tanja; Granado, José; Guitton, Marie-Christine; Hohe, Annette; Holtorf, Hauke; Lucht, Jan M; Rensing, Stefan A; Schlink, Katja; Schulte, Julia; Schween, Gabriele; Zimmermann, Susanne; Duwenig, Elke; Rak, Bodo; Reski, Ralf (2002). "High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library". BMC Plant Biology. 2: 6. doi:10.1186/1471-2229-2-6. PMC 117800. PMID 12123528.
^ Mittmann, F; Dienstbach, S; Weisert, A; Forreiter, C (June 2009). "Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism". Planta. 230 (1): 27–37. Bibcode:2009Plant.230...27M. doi:10.1007/s00425-009-0922-6. PMID 19330350. S2CID 8345708.
^ Reski, Ralf (1998). "Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics". Trends in Plant Science. 3 (6): 209–10. Bibcode:1998TPS.....3..209R. doi:10.1016/S1360-1385(98)01257-6.
^ Reinhard, Christina; Schween, Gabriele; Reski, Ralf; Hohe, Annette; Egener, Tanja; Lucht, Jan M.; Holtorf, Hauke (2004). "An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens". Current Genetics. 44 (6): 339–47. doi:10.1007/s00294-003-0458-4. PMID 14586556. S2CID 45780217.
^ Strepp, René; Scholz, Sirkka; Kruse, Sven; Speth, Volker; Reski, Ralf (1998). "Plant Nuclear Gene Knockout Reveals a Role in Plastid Division for the Homolog of the Bacterial Cell Division Protein FtsZ, an Ancestral Tubulin". Proceedings of the National Academy of Sciences of the United States of America. 95 (8): 4368–4373. Bibcode:1998PNAS...95.4368S. doi:10.1073/pnas.95.8.4368. JSTOR 44902. PMC 22495. PMID 9539743.
^ Koprivova, Anna; Stemmer, Christian; Altmann, Friedrich; Hoffmann, Axel; Kopriva, Stanislav; Gorr, Gilbert; Reski, Ralf; Decker, Eva L. (2004). "Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans". Plant Biotechnology Journal. 2 (6): 517–23. doi:10.1111/j.1467-7652.2004.00100.x. PMID 17147624. S2CID 4645132.
^ BASF and Freiburg University to collaborate on plant biotechnology