Knockout moss

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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-E: Different mutants. Physcomitrella knockout mutants.JPG
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–E: Different mutants.

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.

Contents

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]

Related Research Articles

Gene knockouts are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs.

<span class="mw-page-title-main">Moss</span> Division of non-vascular land plants

Mosses are small, non-vascular flowerless plants in the taxonomic division Bryophytasensu stricto. Bryophyta may also refer to the parent group bryophytes, which comprise liverworts, mosses, and hornworts. Mosses typically form dense green clumps or mats, often in damp or shady locations. The individual plants are usually composed of simple leaves that are generally only one cell thick, attached to a stem that may be branched or unbranched and has only a limited role in conducting water and nutrients. Although some species have conducting tissues, these are generally poorly developed and structurally different from similar tissue found in vascular plants. Mosses do not have seeds and after fertilisation develop sporophytes with unbranched stalks topped with single capsules containing spores. They are typically 0.2–10 cm (0.1–3.9 in) tall, though some species are much larger. Dawsonia, the tallest moss in the world, can grow to 50 cm (20 in) in height. There are approximately 12,000 species.

<span class="mw-page-title-main">Archegonium</span> Organ of the gametophyte of certain plants, producing and containing the ovum

An archegonium, from the Ancient Greek ἀρχή ("beginning") and γόνος ("offspring"), is a multicellular structure or organ of the gametophyte phase of certain plants, producing and containing the ovum or female gamete. The corresponding male organ is called the antheridium. The archegonium has a long neck canal or venter and a swollen base. Archegonia are typically located on the surface of the plant thallus, although in the hornworts they are embedded.

<span class="mw-page-title-main">Protoplast</span> Cell stripped of cell-wall

Protoplast, is a biological term coined by Hanstein in 1880 to refer to the entire cell, excluding the cell wall. Protoplasts can be generated by stripping the cell wall from plant, bacterial, or fungal cells by mechanical, chemical or enzymatic means.

Pharming, a portmanteau of "farming" and "pharmaceutical", refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO). Pharming is also known as molecular farming, molecular pharming or biopharming.

Polycomb-group proteins are a family of protein complexes first discovered in fruit flies that can remodel chromatin such that epigenetic silencing of genes takes place. Polycomb-group proteins are well known for silencing Hox genes through modulation of chromatin structure during embryonic development in fruit flies. They derive their name from the fact that the first sign of a decrease in PcG function is often a homeotic transformation of posterior legs towards anterior legs, which have a characteristic comb-like set of bristles.

<i>Physcomitrella patens</i> Species of moss

Physcomitrella patens is a synonym of Physcomitrium patens, the spreading earthmoss. It is a moss, a bryophyte used as a model organism for studies on plant evolution, development, and physiology.

<span class="mw-page-title-main">Gene targeting</span> Genetic technique that uses homologous recombination to change an endogenous gene

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism. It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombination. Gene targeting can be used to make a range of sizes of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much smaller changes to the existing DNA such as a single base-pair change. Gene targeting relies on the presence of a repair template to introduce the user-defined edits to the DNA. The user will design the repair template to contain the desired edit, flanked by DNA sequence corresponding (homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular genomic region. In this way Gene Targeting is distinct from natural homology-directed repair, during which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA. The alteration of DNA sequence in an organism can be useful in both a research context – for example to understand the biological role of a gene – and in biotechnology, for example to alter the traits of an organism.

<span class="mw-page-title-main">Genetically modified plant</span> Plants with human-introduced genes from other organisms

Genetically modified plants have been engineered for scientific research, to create new colours in plants, deliver vaccines, and to create enhanced crops. Plant genomes can be engineered by physical methods or by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors. Many plant cells are pluripotent, meaning that a single cell from a mature plant can be harvested and then under the right conditions form a new plant. This ability is most often taken advantage by genetic engineers through selecting cells that can successfully be transformed into an adult plant which can then be grown into multiple new plants containing transgene in every cell through a process known as tissue culture.

<span class="mw-page-title-main">Ralf Reski</span> Plant biologist

Ralf Reski is a German professor of plant biotechnology and former dean of the Faculty of Biology of the University of Freiburg. He is also affiliated to the French École supérieure de biotechnologie Strasbourg (ESBS) and Senior Fellow at the Freiburg Institute for Advanced Studies.

<i>Ceratodon purpureus</i> Species of moss

Ceratodon purpureus is a dioicous moss with a color ranging from yellow-green to red. The height amounts to 3 centimeters. It is found worldwide, mainly in urban areas and next to roads on dry sand soils. It can grow in a very wide variety of habitats, from polluted highway shoulders and mine tailings to areas recently denuded by wildfire to the bright slopes of Antarctica. Its common names include redshank, purple forkmoss, ceratodon moss, fire moss, and purple horn toothed moss.

<span class="mw-page-title-main">Somatic fusion</span> Genetic modification fusing plants into a hybrid

Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between different varieties of the same species or between two different species.

Dehydrin (DHN) is a multi-family of proteins present in plants that is produced in response to cold and drought stress. DHNs are hydrophilic, reliably thermostable, and disordered. They are stress proteins with a high number of charged amino acids that belong to the Group II Late Embryogenesis Abundant (LEA) family. DHNs are primarily found in the cytoplasm and nucleus but more recently, they have been found in other organelles, like mitochondria and chloroplasts.

<span class="mw-page-title-main">International Moss Stock Center</span>

The International Moss Stock Center (IMSC) is a biorepository which is specialized in collecting, preserving and distributing moss plants of a high value of scientific research. The IMSC is located at the Faculty of Biology, Department of Plant Biotechnology, at the Albert-Ludwigs-University of Freiburg, Germany.

<span class="mw-page-title-main">Moss bioreactor</span>

A moss bioreactor is a photobioreactor used for the cultivation and propagation of mosses. It is usually used in molecular farming for the production of recombinant protein using transgenic moss. In environmental science moss bioreactors are used to multiply peat mosses e.g. by the Mossclone consortium to monitor air pollution.

In molecular biology mir-529 microRNA is a short RNA molecule. MicroRNAs function to regulate the expression levels of other genes by several mechanisms.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

Circadian Clock Associated 1 (CCA1) is a gene that is central to the circadian oscillator of angiosperms. It was first identified in Arabidopsis thaliana in 1993. CCA1 interacts with LHY and TOC1 to form the core of the oscillator system. CCA1 expression peaks at dawn. Loss of CCA1 function leads to a shortened period in the expression of many other genes.

<span class="mw-page-title-main">Phytoglobin</span>

Phytoglobins are globular plant proteins classified into the globin superfamily, which contain a heme, i.e. protoporphyrin IX-Fe, prosthetic group. The earliest known phytoglobins are leghemoglobins, discovered in 1939 by Kubo after spectroscopic and chemical analysis of the red pigment of soybean root nodules. A few decades after Kubo's report the crystallization of a lupin phytoglobin by Vainshtein and collaborators revealed that the tertiary structure of this protein and that of the sperm whale myoglobin was remarkably similar, thus indicating that the phytoglobin discovered by Kubo did indeed correspond to a globin.

References

  1. 1 2 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. S2CID   8345708.
  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. S2CID   45780217.
  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