The chicken B cell line DT40: a novel tool for gene disruption experiments

Journal of Immunological Methods 249 (2001) 1–16 www.elsevier.nl / locate / jim

Recombinant Technology

The chicken B cell line DT40: a novel tool for gene disruption experiments Pernille Winding, Martin W. Berchtold* Institute of Molecular Biology, Øster Farimagsgade 2 A, DK-1353, Copenhagen K, Denmark Received 10 September 2000; received in revised form 16 November 2000; accepted 16 November 2000

Abstract The use of the chicken DT40 B cell line is increasing in popularity due to the ease with which it can be manipulated genetically. It offers a targeted to random DNA integration ratio of more than 1:2, by far exceeding that of any mammalian cell line. The facility with which knockout cell lines can be generated, combined with a short generation time, makes the DT40 cell line attractive for phenotype analysis of single and multiple gene disruptions. Advantage has been taken of this to investigate such diverse fields as B cell antigen receptor (BCR) signaling, cell cycle regulation, gene conversion and apoptosis. In this review, we give a historical introduction and a practical guide to the use of the DT40 cell line, along with an overview of the main topics being researched using the DT40 cell line as a model system. These topics include B cell-specific subjects such as B cell signaling and Ig rearrangement, and subjects common to all cell types such as apoptosis, histones, mRNA modification, chromosomal maintenance and DNA repair. Attention is in each case brought to peculiarities of the DT40 cell line that are of relevance for the subject. Novel applications of the cell line, e.g., as a vector for gene targeting of human chromosomes, are also discussed in this review.  2001 Elsevier Science B.V. All rights reserved. Keywords: DT40 chicken B cell line; Homologous recombination; Gene targeting; B cell signaling; Apoptosis; Ig rearrangements

Abbreviations: 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; ALV, avian leukosis virus; ARS, autonomously replicating sequence; ATM, ataxia telangiectasia mutated; BCR, B cell antigen receptor; DNA-PK, DNA-dependent protein kinase; EST, expressed sequence tag; HDAC, histone deacetylase; HR, homologous recombination; IgM, immunoglobulin of M isotype; IP3 R, inositol 3-phosphate receptor; ITAM, immunoreceptor tyrosine-based activation motifs; ITIM, immunoreceptor tyrosinebased inhibition motifs; LTR, viral long terminal repeat; MAPK, mitogen-activated protein kinase; NHEJ, non-homologous endjoining; PAP, poly(A) polymerase; PKC, protein kinase C; PLCg2, phospholipase C, type g2; PLD, phospholipase D; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; RTPCR, reverse transcription-polymerase chain reaction; YAC, yeast artificial chromosome *Corresponding author. Tel.: 145-35-322-089; fax: 145-33935-220. E-mail address: [email protected] (M.W. Berchtold).

1. Introduction The DT40 chicken B cell line is derived from an avian leukosis virus (ALV)-induced bursal lymphoma. The ALV was injected intravenously into Hyline SC chickens and a resulting tumor was twice transplanted in vivo prior to cell culturing (Baba and Humphries, 1984; Baba et al., 1985). The DT40 cells are small, approximately 10 mm in diameter, and have a high nucleus to cytoplasm ratio (Fig. 1). They carry IgM on the surface, and although Baba et al. (1985) did not find evidence of secreted IgM, others have (Takagaki et al., 1996; Takami et al., 1999). DT40 cells do release ALV into the surroundings,

0022-1759 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 00 )00333-1

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Fig. 1. Transmission electron microscopy of proliferating (A) and apoptotic (B) DT40 cells. The cells were grown under the described conditions and apoptosis was induced for 18 h with 1 mg / ml ionomycin. ER, endoplasmatic reticulum; M, mitochondria; N, nucleus; Nu, nucleolus; V, viral particles. The scale bar in (A) equals 1 mm.

but in negligible amounts (Baba et al., 1985). In keeping with the observation that nearly 85% of all ALV-induced tumors are disrupted in the c-myc locus, the DT40 cells have integrated the 39 viral long terminal repeat (LTR) upstream of the c-myc gene (Hayward et al., 1981). The 39 LTR is in the same orientation as c-myc, elevating the transcription levels 100-fold in this cell line compared with normal avian bursal cells (Baba et al., 1985). Whereas Baba et al. (1985) detected a normal and an altered c-myc allele, Kim et al. (1990) reported a possible mitotic recombination event among the parental alleles, as they describe the loss of the normal c-myc allele and instead find two copies of the altered c-myc gene. DT40 cells continue to undergo gene conversion, as seen by testing for the presence or absence of various restriction sites within the light chain variable region (Thompson et al., 1987). Although the DT40 cell line may best be characterized as being a bursal B cell line, the ongoing gene conversion and the fact that v-myc transformed cells can reconstitute an ablated bursa of Fabricius suggests, that the DT40 cell line is arrested at a bursal stem cell stage of differentiation (Baba et al., 1985; Thompson et al., 1987; McCormack et al., 1991).

2. Methods

2.1. Propagation and standard techniques DT40 cells can be propagated as other vertebrate / mammalian cell lines. Optimal growth conditions are in RPMI 1640 with L-glutamine (Gibco-BRL), 10% FCS, 1% chicken serum, 2 mM L-glutamine and 10–100 mM b-mercaptoethanol; addition of penicillin and streptomycin to 100 u / l is optional. The FCS and chicken serum should be heat inactivated at 568C for 30 min to prevent the B cell IgM from activating complement. When cultured at 408C with 5% CO 2 , the generation time for wild-type cells is about 10 h. Lower temperatures, e.g., 378C, are also applicable, but lead to an increase in the generation time. Due to the short generation time, care should be taken not to overgrow or starve the cells. DT40 cells grow in a semi-detached fashion; trypsination is not necessary for recovery of the cells. Addition of DMSO to 10% makes it possible to freeze the cells and store them at 2808C or below. The DT40 cells are readily transfected by electroporation and DNA, RNA and protein extraction protocols used for mammalian cell lines are directly employable. An important feature of this cell line is the B cell receptor (BCR)

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signaling pathway, which, if it is not of direct research interest, can be used to induce and study apoptosis. This can be achieved by engaging the BCR with the anti-chicken IgM monoclonal antibody M4 (1–4 mg / ml), which is commercially available (Southern Biotechnology Associates, Birmingham, AL) (Chen et al., 1982; Takata et al., 1995). DT40 cells are also apoptotically responsive to radiation, osmotic, oxidative and nutritional stresses (Lahti, 1999).

2.2. Gene disruption The most exceptional feature of the DT40 cell line is the high ratio of targeted to random DNA integration. This was first reported by Buerstedde and Takeda (1991), who transfected the cell line with different constructs, and found that targeted integration does not depend upon integration into the light chain locus, nor that the locus targeted needs to be transcribed. This phenomenon was furthermore found only to apply for early lineage chicken B cell lines and not other chicken cell lines (Buerstedde and Takeda, 1991). How DT40 cells so readily integrate DNA by homologous recombination is not yet clear. Disruption of RAG-2, a recombination activating gene, had no effect on the frequency of targeted integration (Takeda et al., 1992). On the other hand, disruption of Rad54, a gene believed to be involved in double strand break repair of DNA, reduced the targeted integration frequency by two orders of magnitude (Bezzubova et al., 1997). DNA repair gene products may thus be involved in homologous recombination, but this may be specific for chicken cells (Bezzubova et al., 1997). Additional factors that aid the construction of a DT40 knockout cell line are that chicken introns typically are small and therefore genomic DNA can be amplified by PCR, and that chicken genomic and cDNA libraries are commercially available (Stratagene and Clontech). The chicken codon usage is described in Nakamura et al. (2000), which may be helpful when designing degenerate primers. A newly created expressed sequence tag (EST) database with over 7000 ESTs from bursal lymphocytes will further facilitate finding gene disruption targets in DT40 cells (Abdrakhmanov et al., in press). One point of consideration is that, although most genes

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have two alleles in DT40 cells, cases have been described where only 1 (Syk), and more arduously 3 (Lyn) and 4 (Cbl) gene targeting events were required to disrupt the gene in question (Takata et al., 1994; Yasuda et al., 2000). Generally, a knockout construct for DT40 cells should contain between 2 and 5 kb homologous flanking region on either side of a marker gene that confers a drug resistance. The marker gene can either be inserted into the sequence or replace part of the sequence. Usually, the drug resistance cassette is placed in reverse orientation of the direction of transcription of the gene to be disrupted and the downstream homologous flanking region should be cloned so that it is out of frame with the upstream flanking region of the construct. The construct must be linearized prior to electroporation (550 V, 25 mF, BioRad Genepulser II, 0.4 cm cuvette) of 10 7 cells with 10–25 mg DNA. The fastest way of selecting for drug-resistant single cell clones is by transfecting the cells, recovering in 20 ml non-selective media for 24 h, then harvesting and resuspending the cells in 85 ml selective media and aliquoting 200 ml into 96-well plates. Five to 8 days should be allowed for selection. Alternative methods can be found in (Lahti, 1999) or on the DT40 web page (http: / / genetics.hpi.uni-hamburg.de / dt40.html). There are seven available selection markers that confer resistance to: (final concentration in parentheses) blasticidin S (50 mg / ml), bleomycin / Zeocin (300 mg / ml), mycophenolic acid (10–30 mg / ml), histidinol (0.5–1 mg / ml), hygromycin B (1.5–2 mg / ml), neomycin / G418 (2 mg / ml) and puromycin (0.5 mg / ml). Recently, a DT40 cell line utilizing the cre / loxP system for site-specific recombination has been described using a conditionally induced cre recombinase (Fukagawa et al., 1999a). This makes it possible to work without the marker gene, thus making multiple gene disruptions possible. The same paper reports on the construction of a counter selectable marker system in DT40 based on the HPRT gene (Fukagawa et al., 1999a).

3. Research topics The ease with which homologous recombination can be achieved in DT40 cells has resulted in a long

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list of knockout cell lines (Table 1). Since the first publication on homologous recombination in 1991 (Buerstedde and Takeda, 1991), the number of papers describing gene disruptions / knockouts has

increased nearly exponentially. These knockouts have contributed to our understanding of diverse processes including B cell antigen receptor signaling, histone gene function, RNA processing, DNA repair,

Table 1 Deleted and disrupted genes reported in the DT40 cell line Gene

Function

Field

Reference

Lyn Syk Btk

Tyrosine phosphorylation Tyrosine phosphorylation Tyrosine phosphorylation

BCR signaling BCR signaling BCR signaling

BLNK PLC-g2 Csk CD45 SHP-1 SHP-2 SHIP Akt IP3 R I / II / III Shc Grb2 Grap Cbl 01H1 02H1 .10H1 03H1 H1L H1R H2B-V H3-IV/ H3-V HMG-17 HMG-14a 57 kb deletion 110 kb deletion chHDAC-1 / -2 chHDAC-3 CstF-64 ASF / SF2 RAG-2 Rad51 Rad52 Rad54 BLM KU70 MRE11 ATM c-Abl CENP-C Ggccnd1 Annexin 2 Annexin 5

Adaptor protein Phospholipase Tyrosine phosphorylation Tyrosine phosphatase Tyrosine phosphatase Tyrosine phosphatase Inositol phosphatase Protein kinase IP3 receptors / Ca 21 channels Adaptor protein Adaptor protein Adaptor protein Adaptor protein Histone H1 variant Histone H1 variant Histone H1 variant Histone H1 variant Histone H1 variant Histone H1 variant Histone H2B variant Histone H3 variants Non-histone chromosomal protein Non-histone chromosomal protein Half set of histone genes Allele of major histone gene cluster Histone deacetylase Histone deacetylase Polyadenylation factor subunit Splicing factor Recombination factor DNA repair DNA repair DNA repair Helicase subunit of DNA-PK DNA repair DNA repair / cell cycle control Tyrosine phosphorylation Centromeric protein Cyclin D1 Ca 21 binding protein Ca 21 binding protein

BCR signaling BCR signaling BCR signaling BCR signaling BCR signal inhibitor BCR signal inhibitor BCR signal inhibitor BCR signaling Ca 21 flux BCR signaling BCR signaling BCR signaling BCR signal inhibitor Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Chromatin organization Transcription regulation Transcription regulation RNA processing RNA processing Ig rearrangement Chromosome maintenance Chromosome maintenance Chromosome maintenance Chromosome maintenance Chromosome maintenance Chromosome maintenance Chromosome maintenance Chromosome maintenance Chromosome maintenance Cell cycle control Ca 21 flux Ca 21 flux

Takata et al., 1994 Takata et al., 1994 Takata and Kurosaki, 1996; Uckun et al., 1996 Ishiai et al., 1999 Takata et al., 1995 Hata et al., 1994 Yanagi et al., 1996 Ono et al., 1997 Maeda et al., 1998 Ono et al., 1997 Pogue et al., 2000 Sugawara et al., 1997 Hashimoto et al., 1998 Hashimoto et al., 1998 Hashimoto et al., 1998 Yasuda et al., 2000 Seguchi et al., 1995 Takami et al., 2000 Takami et al., 2000 Takami et al., 2000 Takami et al., 2000 Takami et al., 2000 Takami et al., 1995b Takami et al., 1995a Li and Dodgson, 1995 Li et al., 1997b Takami et al., 1997 Takami and Nakayama, 1997a Takami et al., 1999 Takami and Nakayama, 2000 Takagaki and Manley, 1998 Wang et al., 1996 Takeda et al., 1992 Sonoda et al., 1998 Yamaguchi-Iwai et al., 1998 Bezzubova et al., 1997 Wang et al., 2000 Takata et al., 1998 Yamaguchi-Iwai et al., 1999 Takao et al., 1999 Takao et al., 2000 Fukagawa and Brown, 1997 Lahti et al., 1997 Kubista et al., 1999 Kubista et al., 1999

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cell cycle and calcium signaling. A selection of data from some of these papers is detailed below.

3.1. BCR signaling The DT40 cell line is the choice model system for studying BCR signaling due to its compliant genetic system. Much of what is known about the activation and interaction of the multiple components that make up the BCR signaling pathway have been elucidated in this cell line. The DT40 B cell antigen receptor is of the IgM isotype and can be stimulated by antireceptor antibodies to undergo a response similar to that of other B cells. This ultimately leads to apoptosis in DT40 cells, thereby mimicking the

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elimination of self-reactive B cells (Takata et al., 1995). Cross-linking of B cell antigen receptors sets off a chain of events. First numerous tyrosine phosphorylations occur followed by either attenuation or inhibition of the activation signal by phosphatase activities. If the activation signal prevails, several effector pathways transduce the signal via, e.g., an increase in intracellular calcium or activation of MAPK cascades (reviewed by Kurosaki, 1998, 1999, 2000; DeFranco, 1999). An overview of the signaling pathways is presented in Fig. 2.

3.1.1. PTKs Although the first response to BCR activation is tyrosine phosphorylation, the BCR itself does not

Fig. 2. BCR signaling. (A) Activation of BCR signaling by cross-linking of BCRs. (B) Inhibition of BCR signaling. (C–F) Effector pathways activated by stimulation of the BCR. (C) PLC-g2 pathway. (D) PI3-K pathway. (E) Ras pathway. (F) Rac pathway. PTKs are in green, inhibitory molecules are in red, the main molecule of an effector pathway is in blue. All Iga and Igb contain ITAMs (green squares) and FcgRIIB contains an ITIM (red rectangle). Modified after DeFranco (1999).

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have intrinsic tyrosine phosphorylation activity. It therefore relies on an assortment of protein tyrosine kinases (PTKs) and associated molecules. It is through the immunoreceptor tyrosine-based activation motifs (ITAMs) of Iga and Igb, two molecules that are pair-wise associated with the BCR, that the BCR is linked to the signaling machinery (Fig. 2A). The ITAMs each contain one tyrosine residue, which is phosphorylated by the PTK Lyn as the first event during BCR stimulation. This makes a docking site for the SH2 containing PTK Syk, which upon binding to the phosphorylated ITAM is activated by phosphorylation by Lyn or itself (Takata et al., 1994; Kurosaki et al., 1995). In all, three PTKs play a part in DT40 BCR signaling and they each represent one of the three types of non-receptor cytoplasmic PTKs that are found in all B cells: the Src-PTK Lyn, Syk from the Syk-PTK family and the Tec family member Btk (Takata et al., 1994; Takata and Kurosaki, 1996; Uckun et al., 1996). ZAP-70, the other Syk-PTK family member that has been shown to be functionally equivalent to Syk in DT40 Syk 2 cells, is expressed only in T cells (Kong et al., 1995, 1996). Lyn is the only Src-PTK in DT40 cells and it was in Lyn deficient cells that the importance of the SrcPTKs for BCR signaling was demonstrated (Takata and Kurosaki, 1995). Lyn and Syk mediate nearly, if not all, the tyrosine phosphorylations taking place in the cell upon BCR engagement (Takata and Kurosaki, 1996). These two PTKs act together to phosphorylate Btk upon BCR stimulation. Btk is subsequently autophosphorylated, rendering itself fully active (Kurosaki and Kurosaki, 1997). Disruption of Btk does not have a direct effect on the tyrosine phosphorylation pattern in anti-IgM stimulated cells (Takata and Kurosaki, 1996). This demonstrates the primary role of the Lyn and Syk PTKs and the secondary role of Btk in mediating the BCR signal, despite previous reports of Btk being necessary for Syk activation. Upon anti-IgM stimulation of the BCR, Syk phosphorylates the adaptor protein BLNK, making docking sites for PLC-g2 SH2 domains and thereby recruits PLC-g2 to the BCR signaling complex (Fu et al., 1998; Ishiai et al., 1999) (Fig. 2C). Syk and Btk then phosphorylate PLC-g2, activating the phospholipase (Takata et al., 1994; Takata and Kurosaki,

1996). PLC-g2 is the only PLCg in DT40 cells (Takata et al., 1995). It mediates the signal from the BCR by cleaving PI-4,5-P2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3 ). IP3 binds to IP3 receptors on the endoplasmatic reticulum leading to a rapid and high increase in intracellular Ca 21 concentration. This signal is inhibited in PLCg2 disrupted cells (Takata et al., 1995). Syk and Btk also induce phospholipase D (PLD) activity through activation of PLC-g2 in DT40 cells (Hitomi et al., 1999). PLC-g2 disrupted cells are blocked in apoptosis perhaps because PLD may be important for transducing proliferative responses. PLD may have this role, as inhibition of PLD induces apoptosis in DT40 cells (Hitomi et al., 1999). Downstream of PLC-g2 is protein kinase Cm (PKCm), a serine / threonine kinase that is associated with the BCR complex. PKCm may be activated by DAG and functions as a negative feedback element inhibiting phosphorylation of PLC-g2 by Syk, as found by genetic analysis in Syk, Btk, Lyn and PLC-g2 deficient DT40 cells (Sidorenko et al., 1996). Another negative regulator of Syk and Lyn is the tyrosine kinase Csk, which phosphorylates SrcPTKs on a C-terminal negative regulatory tyrosine residue (Fig. 2B). In Csk disrupted cells, both Lyn and Syk are constitutively active, but no downstream events take place; these still require stimulation of the BCR (Hata et al., 1994). As Syk is not a Src-PTK, the increased activity observed for Syk in Csk deficient cells may be due to Lyn (Kurosaki et al., 1994; Li et al., 1997a).

3.1.2. PTPs The role of protein tyrosine phosphatases (PTPs) during BCR signaling is to maintain or inhibit the activation signal that is initiated by the stimulation of the BCR. CD45 is a Src-PTK-specific transmembrane protein tyrosine phosphatase, which is required for normal BCR signaling by dephosphorylating Lyn (Yanagi et al., 1996). The phenotype of CD45 disrupted DT40 cells is much the same as that of Lyn disrupted cells. Both the tyrosine phosphorylation patterns of whole cell proteins and the Ca 21 mobilization profile are alike in these two mutant cell lines. This is most likely due to the hyperphosphorylation of Lyn on the C-terminal negative regulatory

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tyrosine residue. There is no alteration in the activity of Syk in CD45 disrupted cells (Yanagi et al., 1996). Inhibition of the BCR signal occurs when the BCR is cross-linked to the transmembrane Fc receptor, FcgRIIB (Fig. 2B). An immunoreceptor tyrosinebased inhibitory motif (ITIM) on the FcgRIIB cytoplasmic domain is phosphorylated by Lyn as a response to the cross-linking (Maeda et al., 1999). The PTPs SHP-1 and SHP-2 are then called into action by binding to the phosphorylated ITIM (Maeda et al., 1998, 1999). They inhibit mobilization of Ca 21 by dephosphorylating Syk and Btk, as shown for SHP-1 (Maeda et al., 1999). These data were found by using a chimeric molecule consisting of the extracellular domain of FcgRIIB and an intracellular domain of an inhibitory receptor, PIR-B, from natural killer cells. PIR-B binds SHP-1 but not SHIP, an inositol polyphosphate 59-phosphatase which is recruited to FcgRIIB in a manner similar to that of SHP-1 and SHP-2. The roles of SHP-1 and SHIP were investigated by analysis of SHP-1 and SHIP deficient DT40 cell lines (Ono et al., 1997). SHIP may be the primary inhibitory phosphatase involved in BCR signaling as it blocks the pathway leading to apoptosis (Ono et al., 1997). SHIP prevents calcium influx as a consequence of dephosphorylating PIP3 (PI(3,4,5)P3 ) to PIP2 (PI(4,5)P2 ) (Bolland et al., 1998; Okada et al., 1998). This hinders activation of both Btk and PLCg (Hashimoto et al., 1999), as both require docking to PIP3 for activation (Kurosaki and Kurosaki, 1997; Bolland et al., 1998).

3.1.3. Effectors Several effector pathways become activated upon BCR engagement (Fig. 2). One is the generation of PIP3 through the PI 3-kinase pathway, other pathways involve PLC-g2 (in part described above) Ras and Rac. PI 3-kinase is an upstream regulator of the serine / threonine Akt kinase (protein kinase B) during BCR signaling (Fig. 2D). Akt kinase activity requires translocation to PIP3 , and this is inhibited by dephosphorylation of PIP3 by SHIP (Aman et al., 1998). The regulation of Akt in DT40 cells has been investigated by the use of various PTK knockout cell lines (Craxton et al., 1999; Gold et al., 1999). Disruption of more than one allele of Akt itself has

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not been possible; no viable clones with disruptions of both alleles can be obtained (Pogue et al., 2000). Taking into account the fact that the Lyn 2 / 2 cells used by Gold et al. (1999) in fact were Lyn / Syk double disrupted cells (Gold, pers. commun.), the work of Gold et al. (1999) and Craxton et al. (1999) show that at least one of these two PTKs is required for Akt activity and that Lyn alone can mediate transient activation of Akt while Syk is required for maximal and sustained activation of Akt. Syk has previously been shown by Beitz et al. (1999) to be located upstream of PI 3-kinase in this pathway. Btk, on the other hand, is acting downstream of PI 3kinase, due to the necessity of Btk to dock to PIP3 . Two other groups investigating the regulation of Akt also establish Syk as an activator of Akt, but puts Lyn in the role of an inhibitor, as they find a more substantial activation of Akt in Lyn 2 / 2 cells than in wild-type DT40 cells (Li et al., 1999; Pogue et al., 2000). Cross-linking of the BCR causes an increase of cytoplasmic calcium from intracellular stores via activation of PLC-g2 and by capacitative influx through the plasma membrane from the extracellular environment. Calcium signaling, as a response to anti-IgM antibodies and other agonists, has been characterized for wild-type DT40 cells (Kubista et al., 1998, 1999). In DT40 cells all release of calcium from the intracellular stores is governed by the IP3 receptors, IP3 R (Miyakawa et al., 1999). This has been shown in a DT40 mutant disrupted of all IP3 Rs, where neither the ryanodine receptor activator, caffeine, nor IP3 induction lead to release of Ca 21 (Miyakawa et al., 1999). There are three types of IP3 R: type 1, 2 and 3, encoded by separate genes. By making combinations of single and double disrupted cells, it has been possible to investigate the effect of the IP3 Rs on Ca 21 mobilization (Sugawara et al., 1997; Miyakawa et al., 1999). IP3 R-2 is required for proper Ca 21 oscillations and is also the most IP3 sensitive receptor type, whereas IP3 R-1 has the highest dependence for ATP of the three types. In DT40 cells disrupted for all three types of receptors, there is no increase in intracellular calcium at all upon BCR stimulation (Sugawara et al., 1997). The IP3 Rs are, however, not involved in the capacitative influx of calcium, as uptake of calcium through the plasma membrane is unimpeded despite depletion of

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the intracellular stores. Apoptosis is not completely blocked in triple deficient IP3 R cells, indicating that the PKC pathway is also required for this response (Sugawara et al., 1997). However, transcription by NF-AT is completely arrested in triple IP3 R disrupted cells (Sugawara et al., 1997). Another transcription factor, NF-kB, has recently been shown to be regulated by Btk during BCR signaling (Bajpai et al., 2000; Petro et al., 2000). Ras is another effector that becomes activated upon BCR cross-linking and in turn activates ERK — also known as the MAP kinase pathway (Fig. 2E). Activation of Sos, a guanine nucleotide exchange factor, by translocation to the membrane, is a prerequisite for Ras activation. Several adaptor proteins have been implicated for the translocation of Sos, among them Shc, Grb2, the Grb2 homologue Grap and Cbl. After phosphorylation, which requires the activity of both Syk and Lyn, Shc interacts with Grb2, which again forms a complex with Sos (Nagai et al., 1995). There is redundancy among the translocating factors, as ERK activity is only compromised in Grb2 deficient cells and not in Shc or Grap deficient DT40 cells (Hashimoto et al., 1998). PLCg2 is also required for ERK activity after BCR stimulation (Hashimoto et al., 1998). The protooncogene Cbl is a substrate of Lyn and is believed to negatively regulate the Ras and PLC-g2 pathways (Tezuka et al., 1996; Yasuda et al., 2000). Negative regulation of the Ras pathway may be mediated by binding of Cbl to Syk, disabling interaction between the BCR and Syk (Yankee et al., 1999). Similarly inhibition of the PLC-g2 pathway could occur by binding of Cbl to BLNK, inhibiting its association with PLC-g2, as deletion of Cbl in DT40 cells augments the level of BLNK bound PLC-g2 (Yasuda et al., 2000). Activation of ERK requires Syk and Btk, although Btk may be required to sustain the activity rather than to initiate it (Jiang et al., 1998). The requirement of ERK for the functions of Syk and Btk reflects the dependency of ERK activity on their downstream effector PKC, perhaps PCKm (Jiang et al., 1998). As ERK is activated by the GTP-binding protein Ras, JNK and p38 MAPK are activated by the GTP-binding protein Rac1 (Hashimoto et al., 1998) (Fig. 2F). The GTP exchange factor necessary for Rac1 activity is Vav (Kurosaki, 1999). JNK requires

both Syk and Btk PTKs, but not Lyn, as well as intracellular calcium and PKC (Hashimoto et al., 1998; Jiang et al., 1998). DT40 cells deficient in either Syk or Lyn are able to activate p38 MAPK, whereas the double deficient Lyn / Syk mutant cannot (Jiang et al., 1998). p38 MAPK activation takes place in IP3 R deficient cells but not in PLC-g2 disrupted cells, indicating a dependency of PKC (Hashimoto et al., 1998). The co-existence of the three kinases JNK1, p38 MAPK and ERK, that become activated upon BCR engagement in DT40 cells, where they seem redundant, may reflect different fates for B cells within an organism such as proliferation, differentiation and apoptosis.

3.2. Apoptotic stimuli and response Many factors, including drugs, radiation, osmotic and oxidative stress, can induce apoptosis. PTKs have been found to mediate some of the responses to these factors, making the DT40 cell line a valuable tool for dissecting the pathways involved in apoptosis (Fig. 1). Interestingly, disruption of Lyn protects the cells from apoptosis induced by some factors such as UVC radiation (Qin et al., 1997b) and anti-cancer drugs that function as topoisomerase II inhibitors (Maruo et al., 1999). This demonstrates the role of Lyn in pathways initiated by these agents. Syk, on the other hand, protects cells against osmotic stress-induced apoptosis, as induction of this form of apoptosis was enhanced in Syk deficient cells and reduced upon ectopic expression of Syk (Qin et al., 1997a,b). However, functionally active Syk is not required for osmotic stress stimulated tyrosine phosphorylation of cellular proteins, as it is the case for oxidative stress stimulation (Qin et al., 1998b). Oxidative stress induces calcium mobilization, which is inhibited in both Syk and Lyn mutant cell lines (Qin et al., 1996). Syk also protects DT40 cells in an activity independent manner against ceramide (a second messenger in the apoptotic response to cellular stress)-induced apoptosis (Qin et al., 1998a). In the cases where Syk is required but its activity is not, Syk has been hypothesized to function as an adaptor molecule (Qin et al., 1998a). Both Syk and Lyn have also been suggested to mediate X-ray radiation-induced apoptosis, as the activity of each is induced upon radiation (Yang et al., 1995). An

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apoptosis inhibitor, Nr13, which is a Bcl2 cell death antagonist family member, has also been investigated in DT40 cells and was shown to protect the cells from low serum-induced apoptosis (Lee et al., 1999). Electromagnetic fields have been under suspicion of inducing acute lymphoblastic leukemia in children and the DT40 cells have been used as a model system to investigate this. Exposure of DT40 cells to low energy electromagnetic fields has been reported to stimulate the Lyn, Syk, and Btk PTKs and, downstream of these, PLC-g2, resulting in an increase in IP3 levels (Uckun et al., 1995; Dibirdik et al., 1998; Kristupaitis et al., 1998). However, these reports all stem from the same laboratory and have been disputed by other groups, who have found no effect of low energy electromagnetic fields on Btk or on IP3 levels (Miller and Furniss, 1998).

3.3. Ig rearrangements Birds have a mechanism for the differentiation of B cells and diversification of antibody molecules which differs from that of mammals. Birds have a specialized organ, the bursa of Fabricius, which is necessary for diversification and clonal expansion of B cells, and in fact has given name to this cell type. Specific V(D)J rearrangements of the single functional VL and J L genes which yield the light chain and the single functional VH and J H genes, which together with the 15 D H genes, give rise to the heavy chain, takes place only during embryogenesis, prior to colonization of the bursa. This combinatorial diversification is very limited and the major part of the chickens immune repertoire is created by somatic diversification, a process which takes places within the bursa (reviewed by McCormack et al., 1991). DT40 cells have been found to be a good model system for studying this diversification process (Kim et al., 1990). Sequence analysis of 51 DT40 cell clones isolated after 1 year of passage demonstrated that rearrangement of the immunoglobulin light chain gene, specifically of the V segment by V pseudogenes, occurs by gene conversion and nontemplate basepair modifications (Buerstedde et al., 1990; Kim et al., 1990). This does not require the recombination-activating gene, RAG-2 (Takeda et al., 1992). The conversion events have been estimated to take place at an average rate of one event

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per 40 cell divisions (Buerstedde et al., 1990). The error rate of the conversion mechanism is estimated to be low, as measured by the very low proportion of DT40 cells within a culture that do not express surface IgM (Buerstedde et al., 1990).

3.4. Chromatin organization Histones are necessary for chromatin organization in eukaryotes, and are required in large amounts during cell proliferation. There are five classes of histones each represented by a number of subtypes, e.g., there are six genes encoding H1 variants in DT40 cells, each of which has been disrupted (Seguchi et al., 1995; Takami et al., 2000). None of the mutants thus created had any alterations in growth rate or in total amount of expressed histone H1. This is because eukaryotic cells upregulate expression of histone genes to compensate for any losses, thereby maintaining the status quo. In fact, disruption of 10 of the in total 12 alleles of histone H1 had no effect on growth rate nor H1 expression level, although disruption of one additional allele reduced the amount of H1 in the mutant to half normal level (Takami and Nakayama, 1997b). One of the eight H2B genes (Takami et al., 1995b) and two histone H3 genes have been disrupted in DT40 cells (Takami et al., 1995a), none having any pronounced phenotype except an alteration in protein pattern visualized by 2D-PAGE for the H2B mutant. This was also seen for the H1 disrupted mutants, indicating a role for these histones in gene expression (Seguchi et al., 1995; Takami et al., 1995b; Takami and Nakayama, 1997b). Thirty-nine of the 44 histone genes are found in a major histone gene cluster, 110 kb in size, in the chicken genome. Homozygous deletion of either 57 kb or heterozygous deletion of all 110 kb in DT40 cells has proven to have no detrimental effect on proliferation (Takami and Nakayama, 1997a; Takami et al., 1997). This demonstrates the ability of all the histone gene families to compensate for loss of members, a phenomenon found in very diverse organisms.

3.5. RNA modification Histone deacetylases (HDAC) are part of the transcription regulation machinery. By knocking out

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one of the three HDACs in DT40 cells, chHDAC-2, it was found that this gene is involved not only in transcriptional regulation of IgM heavy and light chains, but also in alternative processing of the IgM heavy chain from the mm (membrane) to the ms (secreted) form (Takami et al., 1999). Another alternative processing factor of IgM heavy chain is the polyadenylation factor CstF. CstF-64 is one of the three subunits of CstF, overexpression of which switches m gene expression from the membrane bound to the secreted form in DT40 cells (Takagaki et al., 1996). Disruption of both CstF-64 alleles is lethal, and a gradual decrease in expression of a heterologous gene in a CstF-64 knockout background leads to cell cycle arrest and ultimately to apoptosis, proving the involvement of CstF-64 in cell proliferation (Takagaki and Manley, 1998). The SR (serine / arginine) protein, ASF / SF2, is an essential gene involved in splicing of pre-mRNA (Wang et al., 1996; Mount, 1997). Its mRNA levels are autoregulated as seen by measuring cellular ASF / SF2 mRNA levels, which decrease, while overexpressing the human homologue. This enabled experiments with a tet repressible construct, which gave the first proof that an SR protein can splice specific pre-mRNAs in vivo (Wang et al., 1998). Poly(A) polymerase (PAP) is required for the formation of poly(A) tails on mRNA and is also an essential gene. For unknown reasons it was impossible to knockout the second allele of this gene in DT40 cells, even while heterologously expressing PAP (Zhao and Manley, 1998).

3.6. DNA repair and chromosomal maintenance DT40 cells have a modal chromosome number of 80, with 11 autosomal chromosomes, the ZW sex chromosomes (thus making it a ‘female’) and 67 microchromosomes (Sonoda et al., 1998). This is two more chromosomes than generally found in the chicken (Gallus gallus) and is due to a trisomy of chromosome 2 and an additional microchromosome (Smith and Burt, 1998; Sonoda et al., 1998). Chromosomes are maintained in eukaryotic cells by two modes of DNA repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). Both modes have been investigated in DT40 cells by targeted disruption of genes necessary for either mechanism.

Rad51, Rad52 and Rad54 belong to the RAD52 epistasis group of genes, as defined in Saccharomyces cerevisiae, and function during HR. Rad54 knockout cells are viable, but grow slowly and are X-ray sensitive. Rad52 is involved in genetic recombination as targeted integration frequencies were reduced in the Rad52 disrupted DT40 cell line (Yamaguchi-Iwai et al., 1998). Rad51 deletion is lethal and the only possibility of investigating the disrupted strain is by transfection with a conditionally repressible Rad51 construct (Bezzubova et al., 1997; Sonoda et al., 1998). The Rad51 protein contains a highly conserved ATP-binding domain. However, mutation of the domain and transfection into a Rad51 deficient mutant strain proved that ATP hydrolysis is not needed for proliferation or repair of radiation damaged DNA. ATP hydrolysis is nonetheless, required for targeted integration, which requires DNA binding (Morrison et al., 1999). Disruption of an ATP-dependent helicase, BLM, in DT40 cells leads to enhanced targeted integration and sister chromatid exchange. Disruption of RAD54 in the BLM 2 / 2 cells nearly eliminated targeted integration and reduced the level of sister chromatid exchange (Wang et al., 2000). HR is the mechanism used in sister chromatid exchange, as found when comparing DT40 cells disrupted for Rad51 or Ku70, a subunit of DNA-dependent protein kinase (DNAPK) which is involved in NHEJ (Sonoda et al., 1999). This again indicates that sister chromatid exchange in BLM 2 / 2 cells occur by HR (Wang et al., 2000). Both the HR and NHEJ pathways for DNA repair function in DT40 cells, but where NHEJ is the mechanism of choice during G1 and early S phase, HR is predominantly used during late S and G2 phase (Takata et al., 1998). Two additional gene products involved in HR, Mre11 and ATM, have been investigated in DT40 cells. DT40 cells disrupted for Mre11 accumulate breaks in the chromosomes and ultimately die, proving the essentiality of Mre11 for chromosomal maintenance (Yamaguchi-Iwai et al., 1999). Mre11 functions via HR and its disruption significantly reduces the rate of targeted integration in DT40 cells (Yamaguchi-Iwai et al., 1999). ATM, as Mre11, is involved in repairing double stranded DNA breaks via HR (Morrison et al., 2000). Humans with mutations in the ATM (ataxia telangiectasia mutated)

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gene suffer from multiple disorders that may be related to insufficient cell cycle regulation (Takao et al., 2000). ATM has been shown to interact with c-Abl, which again interacts with p53 (Takao et al., 2000). However, disruption of c-Abl in DT40 cells had no adverse effects and is not essential for ATM function in this cell line (Takao et al., 2000). That c-Abl is obsolete in DT40 cells, could be explained by lack of p53 expression as found by RT-PCR by Takao et al. (1999). On the other hand, ATM also has a p53 independent role in checkpoint control at the G2 / M phase transition, as ATM disrupted DT40 cells do not accumulate at this checkpoint following g-irradiation (Takao et al., 1999). g-Irradiated wildtype DT40 cells always accumulate in G2 / M, apparently lacking a G1 / S phase checkpoint which again could be contributed to the lack of p53 expression (Takao et al., 1999). However, (Tanikawa et al., 2000) have been able to detect p53 protein expression upon treatment of DT40 cells with doxorubicin, a genotoxin, by Western blotting. p53 cannot be detected without the treatment, but this does not exclude a low basal level of expression (Tanikawa et al., 2000). A protein which may be under control of the cell cycle is the conserved centromeric protein, CENP-C. The function of CENP-C, has been investigated by disrupting one allele of the gene and substituting the other with an estrogen receptor fusion gene making its regulation dependable on the presence or absence of 4-hydroxytamoxifen. In this way it was found, that CENP-C is necessary, but not sufficient for the formation of centromeres, and that its structure may be regulated through the cell cycle (Fukagawa and Brown, 1997; Fukagawa et al., 1999b). In the study of chromosomes various methods can be used, for example a chromosome can either be created de novo or an existing chromosome can be manipulated to yield information. DT40 cells can be used as vectors for disruption of genes on human chromosomes. By making chicken / human microcells, it is possible to take advantage of the DT40 cell lines high recombination rate and selectively target a transfected human chromosome for disruption or truncation (Dieken et al., 1996; Kuroiwa et al., 1998; Mills et al., 1999). The rearranged chromosome can subsequently be transferred to mammalian cells for analysis (Dieken et al., 1996; Dieken and

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Fournier, 1996; Koi et al., 1997). Chicken / human microcells are DT40 cells into which one or more human chromosomes have been transferred (Dieken et al., 1996). The targeting efficiency is increased by 1000–10 000-fold (Dieken et al., 1996). A twist to this approach is to target a region on a human chromosome within a DT40 cell for isolation and incorporating that region into a yeast artificial chromosome (YAC). This requires the presence of an ARS (autonomously replicating sequence, a yeast origin of replication), a yeast centromere and a yeast selectable marker on the human chromosome, at the site of interest (Kouprina et al., 1998). These elements can, as described above, be incorporated into the human chromosome by homologous recombination while the chromosome is in a DT40 cell. Upon transforming yeast with total genomic DNA from the DT40 strain, spontaneous circularization occurs between repeated sequences in the human chromosome and YACs of various sizes can be isolated (Kouprina et al., 1998). Ultimately it may be possible to generate mammalian artificial chromosomes (MACs) in DT40 cells in a similar manner (Brown et al., 1996).

4. Conclusions The DT40 cell line is a versatile system, that allows the study of specific signal transduction pathways such as those activated by the BCR or induced by apoptotic factors, as well as the study of modifications, repair and organization of nucleic acids. This cell line has also begun to be used in other fields of biological research. Efforts to elucidate the mechanisms behind cell cycle progression have been taken in DT40 cells by deletion of cyclin D1 (Lahti et al., 1997). The effects of activating a BCR response on migration of B cells subjected to the chemokine stromal cell derived factor-1a (SDF1a) (Guinamard et al., 1999) and the involvement of the PTK Lyn in the proliferative stimulation by granulocyte colony stimulating factor (G-CSF) (Corey et al., 1998; Grishin et al., 2000) have been investigated using the DT40 cell system. The strength of this vertebrate genetic system is that it allows the use of methods that for practical purposes are inconvenient / impossible in mammalian

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systems. These methods include the generation of single and especially multiple knockout cell lines and the reversal of phenotypes brought about by genetic means. Methods that increase the utility of the DT40 system, such as the tet induction system and recently the cre-loxP system, have been established and proven successful in DT40 cells (Wang et al., 1996; Fukagawa et al., 1999a). New applications are already presenting themselves, one example being the use of DT40 cells as vectors for gene targeting or truncation / disruption of human chromosomes or, by taking advantage of the colonizing ability of DT40 cells, it is possible to induce production of human antibodies into egg yolk by injection of the cells into laying hens (Mohammed et al., 1998). Yet a new feature in the DT40 cell line system is the expression of the murine ecotropic retrovirus receptor. The receptor allows infection with retroviruses produced by transfecting commonly used murine retroviral vectors such as pLXSN, pMSCV, pMX and pMXpIE into the BOSC 23 packaging cell line (Krebs et al., 1999). This facilitates the expression of exogenous genes as it is possible to isolate transfectants within 4 days. The advent of these new methodologies suggests that the DT40 cell system will continue to increase in popularity. A good starting point as a new user of the DT40 cell line is to look up the excellent DT40 homepage. The homepage is regularly updated by members of the Buerstedde lab and can be found at: http: / / genetics.hpi.uni-hamburg.de / dt40.html.

Acknowledgements This work was supported by the Danish Research Council and the Danish Cancer Society. We thank Dr. P. Groscurth for the electron microscope pictures and Dr. J. Lahti, Dr. M. Gold and Dr. Z. Gojkovic for critical reading of the manuscript.

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