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Doi:10.1016/s0300-9084(02)00021-4

Techniques for the removal of marker genes from transgenic plants Charles P. Scutt a,*, Elena Zubko b, Peter Meyer b a Reproduction et Développement des Plantes, École Normale Supérieure de Lyon, 46, allée d’Italie, 69364 Lyon cedex 07, France b Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK Received 30 August 2002; accepted 24 October 2002 Abstract
The presence of marker genes encoding antibiotic or herbicide resistances in genetically modified plants poses a number of problems.
Various techniques are under development for the removal of unwanted marker genes, while leaving required transgenes in place. The aim ofthis brief review is to describe the principal methods used for marker gene removal, concentrating on the most recent and promisinginnovations in this technology.
2002 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved.
Keywords: Marker-gene; Antibiotic; Herbicide; Genetically modified organism 1. Introduction
publicity related to the presence of unnecessary marker genesas sufficient reason to warrant their removal.
The addition of genes conferring desired traits to plants In addition to environmental and health concerns, there also requires the inclusion of marker genes that enable the are also practical reasons for the removal of unnecessary selection of transformed plant cells and tissues. These select- marker genes from plants. Both in fundamental and applied able markers are conditionally dominant genes that confer research, there is frequently a need to add two or more the ability to grow in the presence of applied selective agents transgenes to the same plant line. One method for the serial that are toxic to plant cells, or inhibitory to plant growth, such transformation of plants involves the use of two or more as antibiotics and herbicides. Following transformation, the different selectable markers. However, the number of marker continued presence of marker genes in genetically modified genes available is limited and not all of these are well adapted plants usually becomes unnecessary and may also be unde- to all transformable plant species. The combination of sev- sirable. Herbicide resistance marker genes in transgenic cropplants, for example, could escape to wild relatives of the crop eral nuclear transgenes can also be achieved through sexual through the transfer of pollen, potentially leading to the crosses following the transformation of independent plant spread of herbicide resistance in wild plant populations lines. However, this is not possible in plants that must be The presence of antibiotic resistance markers in transgenic propagated by vegetative means. These include non-sexually plants intended for human or animal consumption may also reproducing plants and highly heterozygous varieties whose be a cause for concern. Fears have been expressed that such genetic backgrounds would be greatly changed by sexual genes may be transferred horizontally to microorganisms of reproduction. Examples of such vegetatively propagated the gut flora of man or animals and lead to the spread of plants include: apple, hybrid aspen, banana, cassava, euca- antibiotic resistances in pathogenic microorganisms. Though lyptus, grapevine, potato and strawberries In addition, extensive studies have failed to detect a quantifiable risk of the combination of transgenes by sexual crosses may be slow, this occurrence many biotechnologists view the negative particularly in trees species. The possibility of removingunwanted marker genes following plant transformation al-lows the same marker to be used for the sequential addition offurther transgenes. A second practical reason for the removal * Corresponding author. Tel.: +33-4-72-72-86-03; of marker genes relates to the greater possibility of instability E-mail address: Charlie.Scutt@ens-lyon.fr (C. Scutt) of transgene expression if several homologous marker gene 2002 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved.
PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 0 0 2 1 - 4 C. Scutt et al. / Biochimie 84 (2002) 1119–1126 Table 1Marker genes and selective agents used for plant transformation. cp Can be used for chloroplast transformation Serratia marcescens, Klebsiella Arabidopsis thaliana, Nicotiana tabacum E. coli TN5, Streptoalloteichus hindustanus Klebsiella pneumoniae var. iozaenae Agrobacterium CP4, maize, Petunia copies are present in the same plant Multiple copies of plants, concentrating particularly on the more recent and marker genes could potentially lead to the silencing of the required transgenes through homology-dependent gene si-lencing mechanisms.
The problems associated with the presence of marker 2. Selectable marker genes used for plant
genes in transgenic plants have been known for quite some transformation
time and various studies over the last decade have demon-strated methods for the removal of these, while leaving the Approximately 25 marker genes, mostly conferring resis- desired transgenes in place. All of the methods published up tance to antibiotics or herbicides, have been successfully until recently have suffered from various drawbacks limiting used for plant transformation ). In addition, a num- their efficiency or widespread applicability. Recently, how- ber of so-called marker gene-free approaches to plant trans- ever, a number of studies have presented methods that seem formation have been developed Selection of transformed to offer advantages over earlier techniques. These include tissues in these systems is based on genes that confer the methods for the removal of nuclear marker genes by intrach- ability to proliferate or differentiate in the absence of some romosomal recombination, or using inducible heterologous otherwise essential factor, such as a necessary exogenous recombinases, in addition to novel methods for the removal plant hormone used in tissue culture. The gene that has so far of chloroplast marker genes. The aim of this brief review is to been most widely used in such an approach is the ipt gene describe and compare the different techniques that have been from the Ti plasmid of Agrobacterium tumefaciens, encoding tested for the removal of marker genes from transgenic the enzyme isopentyl transferase This enzyme cata- C. Scutt et al. / Biochimie 84 (2002) 1119–1126 lyzes the synthesis of isopentyl AMP, a precursor of cytoki- sexual crosses with a recombinase-expressing transformant nins. The excessive level of cytokinins produced in plant In either case, both the recombinase gene and its own tissues constitutively expressing the ipt gene leads to a pro- associated marker gene must subsequently be separated from liferation of these tissues on hormone-free media. Plant tis- the desired trait gene by genetic segregation. Two major sues over-expressing the ipt gene exhibit an “extreme shooty problems have been reported that limit the applications of phenotype” characterized by a loss of apical dominance and these simple recombinase systems. Firstly, all of these sys- an inability to produce roots. The removal of the ipt gene can tems require sexual crosses for the removal of recombinase be accomplished using one of the forms of technology for genes and so cannot be used with vegetatively propagated marker gene removal discussed in this review. Recently, ipt plants. Secondly, the expression of microbial recombinases genes of plant origin that also produce elevated cytokinin for prolonged periods in plant cells may result in unwanted levels when over-expressed have been identified through changes to the genome at sites removed from transgene insertions. The use of microbial recombinases for marker A further type of gene that can be specifically of use in gene removal, however, continues in more refined systems advanced strategies for marker gene elimination acts as a such as the MAT and CLX vector systems discussed dominant negative selective marker The proteins en- below. A further advanced use of the Cre-lox recombination coded by such genes act to inhibit the growth of plant tissues system exploits a transformation cassette designed to elimi- in the presence of appropriate selective agents. Under selec- nate multiple tandem insertions of transgenes and to remove tive conditions, these dominant negative markers may be used to identify plant tissues that have lost their marker genesthrough a recombination event brought about by one of the 3.2. Transposable element-based systems techniques discussed in this review. The tms2 gene of Agro-bacterium tumefaciens, e.g., has been used as such a domi- Heterologous plant transposons have also been used for nant negative marker This gene encodes the enzyme the removal of marker genes In one such system, the indolacetamide amidohydrolase that converts napthaline ac- maize Ac transposable element was engineered to contain the etemide (NAM) into the auxin NAA. Plants expressing tms2 ipt gene, conferring a selectable “extreme shooty phenotype” are unable to root on media containing NAM due to elevated The Ac element encodes its own transposase and so its excision conveniently removes this gene along with the iptmarker gene thereby obviating the need for sexualreproduction steps in the procedure. However, transposon- 3. The removal of marker genes from the plant nuclear
based systems of marker gene removal suffer from a number of disadvantages. Their efficiency is low, partly due to thetendency of transposable elements to reinsert elsewhere in 3.1. Simple microbial recombinase-based systems the genome. Excision of transposons is frequently imprecise,and repeated cycles of insertion and excision may lead to the One of the earliest techniques tested for marker gene generation of mutations at numerous unknown loci. The removal involved the heterologous expression of microbial continued presence of heterologous transposons may also recombinase enzymes in plants to excise marker transgenes lead to genomic instability in transgenic plants. For these that were flanked by microbial recombination sequences.
reasons, transposon-based systems seem to be currently less The general method employed for this is illustrated in favored as a means of the removal of marker genes.
. For example, the Cre recombinase enzyme ofbacteriophage P1 has been used to excise marker genes cloned between pairs of 34 bp directly repeated loxP recom-bination sites Such excision events are precise and leave A further conceptually very simple method for marker one loxP site in place. Other microbial recombinase enzymes gene removal is based on the co-transformation of plants that have been similarly used to remove marker genes from using two distinct transgene constructs present in the same transformed plants include the yeast FLP and R recombi- transformed line of A. One of these con- nases The FLP recombinase, encoded by a gene of the structs contains the selectable marker transgene to be used, Saccharomyces cerevisiae 2µ plasmid, catalyzes the recom- while the other includes the desired trait transgene, itself binatorial excision of sequences flanked by directly repeated unlinked to any marker gene. In a variant of this technique, FRT sites. The R recombinase of Zygosaccharomyces rouxii, these two transgenes are inserted into two different T-DNA acts similarly to catalyze recombinatorial excision between elements present in the same “super-binary” plant transfor- mation vector Co-transformation methods for marker In early studies, the introduction of microbial recombi- gene removal are based on the principle that a proportion of nase genes into plant lines carrying desired trait genes was transformed plants carrying the selectable marker gene will achieved by re-transformation of these either with a recom- also have integrated the required trait transgene at a second, binase gene linked to a further selectable marker gene, or by unlinked insertion site. Marker genes can subsequently be C. Scutt et al. / Biochimie 84 (2002) 1119–1126 Fig. 1. Transgene constructions used for the removal of marker genes from transgenic plants. (A) The simple use of microbial recombinases such as Cre, FLPand R (B) An Ac Transposon-based method for the removal of nuclear marker genes (C) The intrachromosomal recombination method for nuclearmarker gene removal (D) The GST-MAT vector system (E) The CRX vector system (F) Removal of chloroplast marker genes by homologousrecombination Ac = maize Activator transposable element, CDS = coding sequence, GFP = gene coding Green Fluorescent Protein. GST promoter =glutathione S-tranferase promoter, ipt = isopentyl transferase gene, LB and RB = left and right Agrobacterium tumefaciens T-DNA border sequences, nptII =neomycin phosphotransferase II gene, TBS = transformation booster sequence, tms2 = indolacetamide amidohydrolase gene, XVE = estrogen-activated hybridtranscriptional regulator gene removed from such plants by genetic segregation. Co- Zubko et al. tested the efficiency of a pair of 352 bp attP transformation methods suffer from the obvious inefficiency regions from bacteriophage k as substrates for ICR in plants.
that only a proportion of plants carrying the selectable During the integration of the k genome into the E. coli marker will also carry the desired trait gene at an unlinked chromosome, the phage k attP region recombines with a site. Furthermore, as for the simple use of heterologous bacterial attB site over a pair of homologous core sequences.
recombination systems, co-transformation methods cannot The process of bacteriophage integration involves a phage- be used for vegetatively propagated plants.
encoded k integrase and a bacterially encoded IntegrationHost Factor (IHF). The construction used for plant transfor- 3.4. An intrachromosomal recombination (ICR) system mation in the studies of Zubko et al. containeda group of three marker and reporter genes flanked by a pair A more recently devised alternative approach to the re- of directly repeated attP sites. This entire element was situ- moval of nuclear transgene markers exploits the natural ated adjacent to a copy of the transformation booster se- nuclear recombination systems present in plants Re- quence (TBS) from Petunia hybrida and a test transgene moval of marker genes by this approach is based on intrach- conferring a desired trait. The TBS has been found to in- romosomal recombination (ICR) between two directly re- crease the frequency of both ICR and illegitimate recombi- peated sequences flanking the marker gene to be excised.
nation events in Petunia, Nicotiana and maize In this C. Scutt et al. / Biochimie 84 (2002) 1119–1126 study, transformed tobacco calli were initially selected on However, in its present form, it does involve a two-stage kanamycin-containing media and subsequently cultured on procedure to select transgenic calli. Calli are transferred from kanamycin-free media to allow for the loss of the nptII gene selective to non-selective media for propagation and then by ICR. The detection of ICR events was based on the re-transferred to a selective shoot-inducing medium to detect acquisition of sensitivity to kanamycin, and confirmed by the (white) tissue that has lost the marker gene. Such lengthy loss of a negative selection tms2 gene marker. Two identical propagation may increase the risk of somaclonal mutations excision events from 11 initial transformed callus cultures In addition, it has been pointed out that the activity of were recovered, in which a 5.9 kb region containing the three attP sequences as recombination substrates has yet to be marker genes and precisely one of the two attP sites had been demonstrated in a large range of plant species, and the lost by ICR. Some illegitimate recombination events in sister mechanism by which the recombination of these sequences occurs in plants is not yet fully understood though the ICR events in plants have previously been found to be very recent results of Siebert and Puchta may go some way rare, with only 10 such events detectable in all of the cells of a 6-week old tobacco plant In the studies of Zubko et al.
however, the use of attP sequences and the TBS seems to have greatly increased the frequency of ICR events, de-spite the absence of the enzymes and co-factors necessary for The MAT (multiautotransformation) vector system repre- the recombination of attP sites in the phage k system. The sents a highly sophisticated approach for the removal of structure of the attP site may partially explain its apparent nuclear marker genes In this system, a chosen trait recombination-stimulating activity, as sequences containing transgene is placed adjacent to a multigenic element flanked a high A + T base composition have been found to favor both by RS recombination sites A copy of the selectable ICR and illegitimate recombination events in plants ipt gene from A. tumefaciens is inserted between these re- However, a further possible explanation for these results has combinase sites, together with the yeast R recombinase gene recently emerged from a study of repair to double-stranded and this entire assembly is situated within a T-DNA element DNA breaks in plants. Such DNA breaks were previously for the Agrobacterium-mediated transformation of plant tis- known to be repaired by recombination events, though this sues. The MAT vector system allows the removal of the R was thought to occur predominantly by illegitimate, rather recombinase gene along with the ipt gene. The system does than by homologous recombination. Siebert and Puchta not, therefore, require any sexual crosses for the removal of however, devised a system capable of measuring the relative marker or recombinase genes, and recombinase expression in frequencies of repairs to double-stranded breaks by homolo- plant tissues is limited to a minimal period of time, thereby gous and illegitimate recombination mechanisms. In this reducing the possibility of any unwanted recombination ef- study, pairs of double-stranded DNA breaks were generated fects. In an earlier version of the MAT vector, R recombinase in a plant transgene insertion by the transient expression of a activity was constitutively up-regulated by the action of the rare-cutting restriction enzyme. In the transgene insertion CaMV 35S promoter. This system was found to incur a risk used, a pair of rare restriction sites to be cut was flanked by of marker gene excision before the selection of transformed partial sequences of the uidA (GUS) reporter gene, of which plant tissues could take place. To circumvent this problem, a the central portion formed a pair of direct repeats. The induc- more recent version of the MAT vector allows for a tion of pairs of double-stranded DNA breaks led to the loss of delay in the excision of the ipt and R recombinase genes. This a marker gene situated between the two rare restriction sites.
is made possible by the use of a chemically inducible glu- In cases where these breaks were then repaired by homolo- tathione S-transferase promoter from maize to drive R re- gous recombination of the repeated GUS sequences, rather combinase gene expression. Once the positive selection of than by non-homologous end-joining, an active GUS gene transformed plant tissues showing an “extreme shooty phe- was reconstituted. This study found that double-stranded notype” has occurred, the excisive recombination of RS sites, breaks could be repaired either by homologous recombina- leading to a loss of the recombinase and marker genes, is tion or by non-homologus end-joining, and that both of these induced by treatment with the herbicide antidote “Safener”.
events occurred at very high frequencies. The high incidence This two-step procedure using MAT vectors has been suc- of ICR events noted in the studies of Zubko et al. cessfully demonstrated for tobacco and hybrid aspen trans- therefore, might be explained by invoking the involvement of formation, both of which are accomplished using organogen- a double-stranded break repair mechanism whose activity esis for plant regeneration. Plant species for which current was in some way stimulated by the presence of attP se- transformation techniques require regeneration of trans- formed embryos from embryogenic cultures were thought to The ICR method of marker gene removal has the advan- be potentially not amenable to selection using the ipt gene tage of relative simplicity as it does not require the expres- However, it has recently been demonstrated that trans- sion of a heterologous recombinase. In addition, this tech- formed rice plants can be regenerated from embryogenic nique does not require any sexual reproduction steps and cultures by the use of the MAT vector system In this could therefore be used for vegetatively propagated plants.
case, transformed embryos that had lost the ipt marker gene C. Scutt et al. / Biochimie 84 (2002) 1119–1126 but retained the desired transgene were selected directly in a up to 104 copies per cell This considerable amplifica- one-step procedure without the occurrence of an “extreme tion can give a very high level of transgene expression, which may be useful for applications requiring high concentrationsof proteins. Examples of these include the engineering of 3.6. The CLX chemically inducible system drought-resistance, or the production of pharmaceuticals inplanta by molecular farming. The chloroplast genome is In a further highly sophisticated approach to nuclear uniquely transmitted through the female germ line in many marker gene removal, the Cre-lox recombination system has crops, reducing the possibility of transgene escape via polli- been engineered to be chemically inducible Antibiotic nation into local wild populations of plants. Examples of selection using the CLX vector system for plant transforma- chloroplast transgenes used to date include: the Cry gene, tion and marker gene removal is based on an nptII gene encoding Bacillus thuringiensis (Bt) toxin to confer insect driven by a constitutive promoter. This nptII gene is resistance and the hST gene encoding human somatatro- positioned adjacent to a Cre-recombinase gene driven by the hybrid, chemically inducible OLexA-46 promoter, and a hy- Transformation of chloroplasts is currently performed by brid XVE gene encoding the binding protein necessary biolistic methods. Following the integration of transgenes for the induction of Cre gene transcription These into the chloroplast genome, a heterogeneous population of three transcription units, with the exception of the constitu- plastids will exist in transformed tissues, and selection using tive promoter driving XVE gene expression, are flanked by a a marker gene is required to produce homoplasmic plants in pair of directly repeated loxP sites. Background expression which the modified plastid genome has completely replaced of the Cre gene in Agrobacterium is avoided by the incorpo- the unmodified one. Transformation cassettes used for chlo- ration of a plant intron. Following the Agrobacterium- roplast transformation contain sequences homologous to two mediated transformation of Arabidopsis root tissues using adjacent regions of the chloroplast genome to allow the the CLX vector system and the selection of transformed integration of the transgenes by homologous recombination.
tissues on kanamycin-containing media, Cre recombinase Selectable marker genes and desired trait transgenes are placed between these homologous recombination sequences.
b-estradiol. As a result of Cre recombinase activity, the XVE The removal of marker genes from the chloroplast genome is coding sequence and the Cre and nptII genes were lost by particularly important as their very high copy numbers could precise excisive recombination between the loxP sites. This otherwise lead to high levels of unwanted marker gene prod- excision led to the close juxtaposition of the promoter previ- ucts. A further argument for the removal of chloroplast ge- ously driving XVE expression, and a previously promoter- netic markers relates to the conservation of activity that often less Green Fluorescent Protein (GFP) coding sequence, with exists between chloroplast and bacterial promoters. This concomitant activation of GFP expression. As with the MAT could increase the risk of the horizontal transfer of functional vector system, the use of the CLX vector for transformation marker genes from plants to bacteria. Fewer resistance genes and marker gene removal exposes plants to recombinase are available for chloroplast than for nuclear transformation, activity for the minimum possible time period, and does so with most of the published studies based on the use of the after an adequate period of time has elapsed to permit trans- aadA gene The paucity of available selection formant selection. The CLX vector system benefits also from methods for chloroplast transformation further increases the a particularly tightly regulated system of chemical induction value of technology that enables the recycling of marker The procedure could be used for vegetatively propa- genes for the serial re-modification of a single transgenic gated species and may be particularly well adapted to crop species requiring transformation by the regeneration of em-bryo cultures.
4.1. Homologous recombination systems As the integration of foreign transgenes into the chloro- 4. The removal of marker genes from the chloroplast
plast genome takes place by homologous recombination, it was entirely logical to test this native plant mechanism as ameans for the removal of marker genes from the chloroplast The genetic modification of chloroplasts can represent an The first demonstration of this technique was attractive alternative to engineering of the plant nuclear ge- performed on the unicellular green alga, Chlamydomonas nome for some applications Unlike the nuclear transfor- reinharIamtham and Day then demonstrated its mation of higher plants, chloroplast transformation takes applicability to higher plants using a construction of three place almost invariably by homologous recombination, re- marker genes which shared two identical promoter se- sulting in precise and predictable genetic modifications. The quences of 174 bp and three identical terminator sequences plastid genome is present in multiple copies in each or- of 418 bp. Several different recombinative excision events ganelle, and these can multiply to large numbers, particularly were detected between the similar sets of promoter or termi- in leaf tissues, such that a chloroplast transgene can exist in nator sequences in the series of transgenic plants analyzed in C. Scutt et al. / Biochimie 84 (2002) 1119–1126 these studies. After the removal of antibiotic selection, these already exist. It seems highly likely that continued work in excision events accumulated to high frequency, leading to a this area will soon remove the question of unwanted marker homoplastic, marker-free state in approximately 25% of genes from the debate concerning the public acceptability of transgenic lines in the next generation. Homoplastic marker- transgenic crop plants. The techniques for marker gene re- free plants may be identified in this technique by PCR or by moval under development will also facilitate the more pre- cise and subtle engineering of the plant genome, with wide-spread applications in both fundamental research and 4.2. Cre-lox recombination-based systems Two recent studies have demonstrated that the Cre-lox system can also be used for the removal of plastid transgene Acknowledgements
markers These systems function essentially as forthe removal of nuclear transgenes by Cre-lox recombination.
C.P.S. is funded as a researcher of the Centre National de A Cre-recombinase gene is expressed from a plant transfor- la Recherche Scientifique (CNRS). The laboratory of Repro- mation cassette integrated into the nuclear genome, while an duction et Développement des Plantes (RDP) is funded N-terminal chloroplast-directing signal sequence routes the jointly by the CNRS, the Institut National de la Recherche Cre recombinase protein that is produced to the plastids.
Agronomique, the École Normale Supérieure de Lyon and Plastid transgene constructions for use with these methods of the Université Claude Bernard-Lyon.
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Source: http://www.ens-lyon.fr/RDP/IMG/pdf/scutt02biochim.pdf

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HCTZ, HydroDIURIL®, Oretic®, and others), metolazone (Mykrox® and Zaroxolyn®). No known contraindications for Phytase. WARNINGS NDC 54799-917-05 Women who are pregnant, nursing or may become pregnant should consult their physician before using this product. Brief Summary of Prescribing Information PRECAUTIONS DESCRIPTION Zinc supplements may cause mild nausea in susc

4suppdatera.se

APPLICATION FORM STUDENT INFORMATION (Please write in capital letters!) First Name(s) (please underline name used) GUARDIAN(S) INFORMATION Guardian 1 Guardian 2  Please tick if you are the child’s only guardian Square d'Argenteuil 5, B-1410 Waterloo, Belgium STUDENT INFORMATION Has the student had any type of learning support p

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