CRISPR Deletion of miR-27 Impacts Recombinant Protein Production in CHO Cells.

MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of target genes. In that regard, their utility has been demonstrated as a strategy to improve the cellular phenotypes important in the biomanufacturing of recombinant proteins. Common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, both of which require the introduction and expression of extra genetic material in the cell. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate CHO cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in influencing CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We show that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures, making it a potentially interesting target to improve bioprocess performance of CHO cells.

A complete workflow for single cell mtDNAseq in CHO cells, from cell culture to bioinformatic analysis.

Chinese hamster ovary (CHO) cells have a long history in the biopharmaceutical industry and currently produce the vast majority of recombinant therapeutic proteins. A key step in controlling the process and product consistency is the development of a producer cell line derived from a single cell clone. However, it is recognized that genetic and phenotypic heterogeneity between individual cells in a clonal CHO population tends to arise over time. Previous bulk analysis of CHO cell populations revealed considerable variation within the mtDNA sequence (heteroplasmy), which could have implications for the performance of the cell line. By analyzing the heteroplasmy of single cells within the same population, this heterogeneity can be characterized with greater resolution. Such analysis may identify heterogeneity in the mitochondrial genome, which impacts the overall phenotypic performance of a producer cell population, and potentially reveal routes for genetic engineering. A critical first step is the development of robust experimental and computational methods to enable single cell mtDNA sequencing (termed scmtDNAseq). Here, we present a protocol from cell culture to bioinformatic analysis and provide preliminary evidence of significant mtDNA heteroplasmy across a small panel of single CHO cells.

Enhancing recombinant protein and viral vector production in mammalian cells by targeting the YTHDF readers of N6 -methyladenosine in mRNA.

N6 -methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and has been implicated in a wide range of fundamental cellular processes. This modification is regulated and interpreted by a set of writer, eraser, and reader proteins. To date, there have been no reports on the potential of mRNA epigenetic regulators to influence recombinant protein expression in mammalian cells. In this study, the potential of manipulating the expression of the m6A YTH domain-containing readers, YTHDF1, 2 and 3 to improve recombinant protein yield based on their role in regulating mRNA stability and promoting translation were evaluated. Using siRNA-mediated gene depletion, cDNA over-expression, and methylation-specific RNA immunoprecipitation, it is demonstrated that (i) knock-down of YTHDF2 enhances (~2-fold) the levels of recombinant protein derived from GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2 depletion on transgene expression is m6A-mediated; and (iii) YTHDF2 depletion, or over-expression of YTHDF1 increases viral protein expression and yield of infectious lentiviral (LV) particles (~2-3-fold) in HEK293 cells. We conclude that various transgenes can be subjected to regulation by m6A regulators in mammalian cell lines and that these findings demonstrate the utility of epitranscriptomic-based approaches to host cell line engineering for improved recombinant protein and viral vector production.

Reinventing the Wheel: Synthetic Circular RNAs for Mammalian Cell Engineering.

The circular RNA renaissance is upon us. Recent reports demonstrate applications of synthetic circular RNA molecules as gene therapies and in the production of biologics from cell-based expression systems. Circular RNAs are covalently closed loop RNA species that are formed naturally through noncolinear splicing of pre-mRNA. Although once thought to be noncoding artefacts from splicing errors, it is now accepted that circular RNAs are abundant and have diverse functions in gene regulation and protein coding in eukaryotes. Numerous reports have investigated circular RNAs in various diseases, but the promise of synthetic circular RNAs in the production of recombinant proteins and as RNA-based therapies is only now coming into focus. This review highlights reported uses of synthetic circular RNAs and describes methods for generating these molecules.

Investigation and circumvention of transfection inhibition by ferric ammonium citrate in serum-free media for Chinese hamster ovary cells.

While reliable transfection methods are essential for Chinese hamster ovary (CHO) cell line engineering, reduced transfection efficiencies have been observed in several commercially prepared media. In this study, we aimed to assess common media additives that impede efficiency mediated by three chemical transfection agents: liposomal-based (Lipofectamine 2000), polymer-based (TransIT-X2), and lipopolyplex-based (TransIT-PRO). An in-house GFP-expressing vector and serum-free medium (BCR-F12: developed for the purposes of this study) were used to analyze transient transfection efficiencies of three CHO cell lines (CHO-K1, DG44, DP12). Compared to a selection of commercially available media, BCR-F12 displayed challenges associated with transfection in vendor-prepared formulations, with no detection when liposomal-based methods were used, reduced (<3%) efficiency observed when polymer-based methods were used and only limited efficiency (25%) with lipopolyplexes. Following a stepwise removal protocol, ferric ammonium citrate (FAC) was identified as the critical factor impeding transfection, with transfection enabled with the liposomal- and polymer-based methods and a 1.3- to 7-fold increased lipopolyplex efficiency observed in all cell lines in FAC-depleted media (-FAC), although lower viabilities were observed. Subsequent early addition of FAC (0.5-5 hr post-transfection) revealed 0.5 hr post-transfection as the optimal time to supplement in order to achieve transfection efficiencies similar to -FAC medium while retaining optimal cellular viabilities. In conclusion, FAC was observed to interfere with DNA transfection acting at early stages in all transfection agents and all cell lines studied, and a practical strategy to circumvent this problem is suggested.

An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals.

Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.

Continuous translation of circularized mRNA improves recombinant protein titer.

Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A "self-cleavage" peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.

Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance.

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.

Improved yield of rhEPO in CHO cells with synthetic 5' UTR.

The impact of local structure on mRNA translation is not well-defined pertaining to the 5' UTR. Reports suggest structural remodelling of the 5' UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5' UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5' UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer.

Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of ubiquitin carboxyl-terminal hydrolase 14.

miRNAs are potent molecular regulators of cellular behaviour. The manipulation of these small non-coding RNAs has been used to enhance industrially relevant phenotypes in Chinese Hamster Ovary (CHO) cells. We investigated the stable depletion of six miRNAs; miR-204-5p, 338-3p, 378-3p, 409-3p, 455-3p and 505-3p, robustly associated with cell growth rate from a previous profiling study. Inhibition of endogenous miR-378-3p function by miRNA-sponge-decoy improved peak cell density by 59%. Quantitative label free LC-MS/MS proteomic analysis of the fractionated cell cultures at day 4 and 8 of batch culture found 216 cytosolic and 114 membrane-associated proteins differentially expressed with stable miR-378-3p depletion. qRT-PCR of 8 genes; Clic4, Hnrnpa1, Prdx1, Actn4, Usp14, Srxn1, Canx and Gnb1, with unidirectional differential protein expression over the two time points of analysis was carried out. In-silico predictive algorithms; TargetScan and miRDB, were used to decipher possible direct targets of miR-378-3p. The Ubiquitin carboxyl-terminal hydrolase 14 (Usp14) protein was identified in the cytosolic fractions at both timepoints as differentially expressed with an increased abundance of 1.58-fold in the miR-378-3p depleted cells on day 8. Usp14 is a deubiquitinase (DUB) with previous reports of its up-regulation leading to increased proliferation of cancer cells. Overexpression of Usp14 in CHO cells had significant effects on cell growth supporting a role for Usp14 in the increased peak cell density seen with miR-378-3p depletion. This study highlights miR-378-3p as a novel engineering candidate for improving CHO cell growth. The use of subcellular fractionation also improved proteome coverage in the identification of novel miRNA targets.

Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells.

MicroRNAs with their unique ability to target hundreds of genes have been highlighted as powerful tools to improve bioprocess behavior of cells. The common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, which requires the introduction and expression of extra genetic material. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate Chinese hamster ovary (CHO) cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in controlling CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We showed that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures making it a potentially interesting target to improve bioprocess performance of CHO cells.

miR-CATCH Identifies Biologically Active miRNA Regulators of the Pro-Survival Gene XIAP, in Chinese Hamster Ovary Cells.

Genetic engineering of mammalian cells is of interest as a means to boost bio-therapeutic protein yield. X-linked inhibitor of apoptosis (XIAP) overexpression has previously been shown to enhance CHO cell growth and prolong culture longevity while additionally boosting productivity. The authors confirmed this across a range of recombinant products (SEAP, EPO, and IgG). However, stable overexpression of an engineering transgene competes for the cells translational machinery potentially compromising product titre. MicroRNAs are attractive genetic engineering candidates given their non-coding nature and ability to regulate multiple genes simultaneously, thereby relieving the translational burden associated with stable overexpression of a protein-encoding gene. The large number of potential targets of a single miRNA, falsely predicted in silico, presents difficulties in identifying those that could be useful engineering tools. The authors explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH protocol. A biotin-tagged antisense DNA oligonucleotide for XIAP mRNA is designed and used to pull down and capture bound miRNAs. Two miRNAs are chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both results in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increases XIAP protein levels. A 3'UTR reporter assay confirms miR-124-3p to be a bona fide regulator of XIAP in CHO-K1 cells. This method demonstrates a useful approach to finding miRNA candidates for CHO cell engineering without competing for the cellular translational machinery.

A proteomic profiling dataset of recombinant Chinese hamster ovary cells showing enhanced cellular growth following miR-378 depletion.

The proteomic data presented in this article provide supporting information to the related research article "Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of Ubiquitin Carboxyl-Terminal Hydrolase 14" (Costello et al., in press) [1]. Control and microRNA-378 depleted CHO DP12 cells were profiled using label-free quantitative proteomic profiling. CHO DP12 cells were collected on day 4 and 8 of batch culture, subcellular proteomic enrichment was performed, and subsequent fractions were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Here we provide the complete proteomic dataset of proteins significantly differentially expressed by greater than 1.25-fold change in abundance between control and miR-378 depleted CHO DP12 cells, and the lists of all identified proteins for each condition.

Conditional Knockdown of Endogenous MicroRNAs in CHO Cells Using TET-ON-SanDI Sponge Vectors.

MicroRNAs (miRNAs) are small, noncoding RNAs of about 22 nucleotides in length and have proven to be useful targets for genetic modifications for desirable phenotype in the biotech industry. The use of constitutively expressed "miRNA sponge" vectors in which multiple, tandem miRNA binding sites containing transcripts are transcriptionally regulated by a constitutive promoter for down regulating the levels of endogenous microRNAs in Chinese hamster ovary (CHO) cells has shown to be more advantageous than using synthetic antisense oligonucleotides. The application of miRNA sponges in biotechnological processes, however, could be more effective, if expression of miRNA sponges could be tuned. In this chapter, we present a method for the generation of stable CHO cell lines expressing a TET-ON-SanDI-miRNA-sponge that is in theory expressed only in the presence of an inducer.

Manipulating MiRNA Expression to Uncover Hidden Functions.

There are a numerous target prediction algorithms that allow users to identify putative targets of their microRNAs (miRNAs) of interest. Although these tools are useful to gain insight into the potential role of miRNAs in regulating cellular processes, physical manipulation of the expression of the miRNA is the most superior way to truly determine the function of the miRNA in the system of interest. This chapter outlines methods to reveal miRNA function by modulating miRNA expression by transient transfection of miRNA mimics and inhibitors, and stable overexpression and reduction of miRNA expression using plasmid overexpression and sponge vectors.

A nonlethal sampling method to obtain, generate and assemble whole blood transcriptomes from small, wild mammals.

The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where nonlethal sampling is the only option. Whole blood has been suggested as a nonlethal sample type that contains a high percentage of bodywide and genomewide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be nonlethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species. In this study, we developed a nonlethal sampling method, the laboratory protocols and a bioinformatic pipeline to sequence and assemble the whole blood transcriptome, using Illumina RNA-Seq, from wild greater mouse-eared bats (Myotis myotis). For optimal results, both ribosomal and globin RNAs must be removed before library construction. Treatment of DNase is recommended but not required enabling the use of smaller amounts of starting RNA. A large proportion of protein-coding genes (61%) in the genome were expressed in the blood transcriptome, comparable to brain (65%), kidney (63%) and liver (58%) transcriptomes, and up to 99% of the mitogenome (excluding D-loop) was recovered in the RNA-Seq data. In conclusion, this nonlethal blood sampling method provides an opportunity for a genomewide transcriptomic study of small, endangered or critically protected species, without sacrificing any individuals.

Re-programming CHO cell metabolism using miR-23 tips the balance towards a highly productive phenotype.

microRNA engineering of CHO cells has already proved successful in enhancing various industrially relevant phenotypes and producing various recombinant products. A single miRNA's ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to be more productive by favoring metabolic channelling into oxidative metabolism. CHO clones depleted of miR-23 using a miR-sponge decoy demonstrated an average ∼three-fold enhanced specific productivity with no impact on cell growth. Using a cell respirometer, mitochondrial activity was found to be enhanced by ∼30% at Complex I and II of the electron transport system. Additionally, label-free proteomic analysis uncovered various potential novel targets of miR-23 including LE1 and IDH1, both implicated in oxidative metabolism and mitochondrial activity. These results demonstrate miRNA-based engineering as a route to re-programming cellular metabolism resulting in increased productivity, without affecting growth.

CHO cell culture longevity and recombinant protein yield are enhanced by depletion of miR-7 activity via sponge decoy vectors.

Improving the efficiency of recombinant protein production by CHO cells is highly desirable as more complex proteins (MAbs, fusion proteins, blood/clotting factors, etc.) go into development and come onto the market. Previous reports have shown that microRNA (miRNA)-7 overexpression arrests the growth of CHO cells and that its depletion increases the proliferation of various cell types. In this study we generated stable CHO clones that overexpressed a miR-7-specific decoy transcript (sponge) downstream of a green fluorescent protein reporter gene. The miR-7 sponge efficiently diverted miR-7 away from its endogenous targets as exemplified by the increased expression of CDC7. Although the sponge effectively sequestered miR-7, it also appeared to protect the bound miRNA sequence from degradation in the cell, as exemplified by the apparent increase in mature miR-7 levels without any change in primary transcription. Phenotypically, CHO clones with sequestered miR-7 displayed improved maximum cell density (40%), significantly improved viability and an almost two-fold increase in yield of secreted protein in a fed-batch culture. These findings demonstrate that miRNA sponge transcripts could potentially be used in cell line development projects to generate producer clones that grow to higher densities and last longer in the bioreactor - thereby improving product yield.

MiR-7 triggers cell cycle arrest at the G1/S transition by targeting multiple genes including Skp2 and Psme3.

MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.

Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds.

BACKGROUND: Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. RESULTS: cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was confirmed via allele-specific transcript analysis across a range of different accessions. Differentially methylated regions were identified adjacent to ATCDC48 and PDE120, which may represent candidate imprinting control regions. Finally, we demonstrate that expression levels of these three genes in vegetative tissues are MET1-dependent, while their uniparental maternal expression in the seed is not dependent on MET1. CONCLUSIONS: Using a cDNA-AFLP transcriptome profiling approach, we have identified three genes, ATCDC48, PDE120 and MS5-like which represent novel maternally expressed imprinted genes in the Arabidopsis thaliana seed. The extent of overlap between our cDNA-AFLP screen for maternally expressed imprinted genes, and other screens for imprinted and endosperm-expressed genes is discussed.