Knockout moss

A knockout moss is a moss plant in which one or more specific genes are deleted or inactivated ("knocked out") by gene targeting. After 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 as the scientist wants to unravel 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.

Wild-type Physcomitrella and knockout mosses: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row; scale bar corresponds to 1 mm) and a close-up (bottom row; scale bar equals 0.5 mm) are shown. A: Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B–D: Different mutants.[1]

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). Basically, a knockout mouse is engineered in the same way. 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). As mosses are haploid organisms, the regenerating moss filaments (protonemata) can be directly assayed for gene targeting within 6 weeks when utilizing PCR-methods.[4]

Examples

Chloroplast division

The first scientific publication about identification of the function of a hitherto unknown gene utilizing knockout moss appeared 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

By multiple gene knockout Physcomitrella plants were engineered, that lack 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 that is used for gene identification.[1][7]

References
  1. 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.
  2. 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. doi:10.1007/s00425-009-0922-6. PMID 19330350.
  3. Reski, Ralf (1998). "Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics". Trends in Plant Science. 3 (6): 209–10. doi:10.1016/S1360-1385(98)01257-6.
  4. 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.
  5. 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.
  6. 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.
  7. BASF and Freiburg University to collaborate on plant biotechnology
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