new system for analyzing line retrotransposition in the chicken dt4

7/30/2019 new system for analyzing LINE retrotransposition in the chicken DT4

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A new system for analyzing LINE retrotransposition in the chicken DT40

cell line widely used for reverse genetics

Hiroshi Honda a, Kenji Ichiyanagi a, Jun Suzuki a, Takao Ono a, Hideki Koyama b,Masaki Kajikawa a, Norihiro Okada a,

a Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-21 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japanb Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama Ci ty University, 641-12 Maioka-cho,

Totsuka-ku, Yokohama, Kanagawa 244-0813, Japan

Received 14 September 2006; received in revised form 14 February 2007; accepted 19 February 2007

Available online 14 March 2007

Abstract

Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse

transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins.

However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the

lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied

the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established

by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells

that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on

the presence of the essential 3 region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their

chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of

LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system

established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.

2007 Elsevier B.V. All rights reserved.

Keywords: Retrotransposon; Host factor; DNA repair; Reverse genetics

1. Introduction

Long interspersed elements (LINEs) are transposable

elements present in a large number of eukaryotic genomes.

The LINE copies are amplified by a copy-and-paste mechanism

of retrotransposition. During the process, the genomic LINEDNA is first transcribed (copied) to RNA; then this RNA is

reverse transcribed (pasted) to cDNA at the site of insertion,

generating a new LINE copy. The copy numbers of LINEs often

reach several thousand to one million per haploid genome. For

example, the human genome contains ~ 900,000 copies of

LINEs, which constitute ~20% of the genome (Lander et al.,

2001). In addition, LINEs are thought to have a large impact onthe evolution of eukaryotic genomes (Kazazian, 2004); hence,

elucidating LINE dynamics and the molecular mechanisms of

LINE retrotransposition is of great importance in understanding

the mechanisms of genome evolution.

LINEs typically encode two open reading frames (ORFs),

ORF1 and ORF2, both of which are required for the LINE

retrotransposition (Feng et al., 1996; Moran et al., 1996). The

ORF1 protein (ORF1p) binds its own RNA to form a

ribonucleoprotein (RNP) complex (Hohjoh and Singer, 1996;

Kolosha and Martin, 1997). In addition, mouse LINE-1 (L1)

ORF1p has a nucleic acid chaperone activity, which is also

Gene 395 (2007) 116124

www.elsevier.com/locate/gene

Abbreviation s: cDNA, complimentary DNA; cytomegalovirus, CMV;

DSBs, double-strand breaks; EGFP, enhanced green fluorescence protein; EN,

endonuclease; FBS, fetal bovine serum; G418R, G418-resistant; LINEs, long

interspersed elements; L1, LINE-1; L2, LINE-2; Neo, neomycin resistance;

ORFs, open reading frames; ORF1p, ORF1 protein; ORF2p, ORF2 protein;

RFI, relative fluorescence intensity; RNP, ribonucleoprotein; RT, reverse

transcriptase; TPRT, target-primed reverse transcription; UTR, untranslated

region. Corresponding author. Tel.: +81 45 924 5742; fax: +81 45 924 5835.

E-mail address: [email protected] (N. Okada).

0378-1119/$ - see front matter 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2007.02.017
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required for L1 retrotransposition (Martin et al., 2005). Thus,

ORF1p may not only be a structural component but also has an

additional function(s) in retrotransposition. The ORF2 protein

(ORF2p) has an endonuclease (EN) and reverse transcriptase

(RT) domain and forms a large RNP complex with the LINE

RNA and ORF1p (Kulpa and Moran, 2006; Matsumoto et al.,

2006). During retrotransposition, the EN domain nicks a targetsite on chromosomal DNA, and the 3 hydroxyl group

generated by the cleavage is subsequently utilized by the RT

domain to prime the reverse transcription of the RNA in the

complex by a mechanism called target-primed reverse tran-

scription (TPRT) (Luan et al., 1993; Cost et al., 2002) (see

Fig. 1). The late stages of LINE retrotransposition (after TPRT)

involve a second DNA cleavage on the target DNA, removal of

the LINE RNA, synthesis of the second (sense) strand cDNA,

and ligation of the LINE cDNA and target DNA. However,

these reactions have not been precisely resolved.

It is anticipated that host-encoded proteins are involved in

these late stages of LINE retrotransposition. For example,Gilbert et al. (2005) have proposed that products of TPRT are

recognized and processed by the host DNA repair machinery.

Indeed, overexpression of the human L1-encoded protein(s) in

cultured cells induces double-strand breaks (DSBs) recognized

by host factors on the chromosomal DNA (Gasior et al., 2006),

suggesting the involvement of the host DNA repair machinery

in L1 retrotransposition. Bioinformatic studies also have

suggested host protein involvement in joining of the LINE

and target DNA (Zingler et al., 2005; Ichiyanagi et al., 2007).

Previous genetic studies (Morrish et al., 2002; Gasior et al.,

2006) identified a few host mutations that affect retrotransposi-

tion efficiency from mutant cell lines generated by random

mutagenesis or established from a human patient. However, amore comprehensive understanding of the roles of host proteins

in LINE retrotransposition will require a systematic study

examining the effects of a number of single host mutations and

combinations of mutations on LINE retrotransposition. To

facilitate such studies, it would be beneficial to identify cell

lines to which gene targeting can be easily applied. DT40 cells,

a chicken B lymphocyte cell line, is one such candidate because

genes of interest can be efficiently knocked out in this cell line

(Buerstedde and Takeda, 1991). Indeed, a variety of mutant

DT40 lines have been produced, including those in which DNA

repair genes are knocked out (Winding and Berchtold, 2001;Yamazoe et al., 2004). Furthermore, because of the relatively

stable phenotype and karyotype of DT40 cells, targeting of

multiple genes is feasible as well, thus facilitating the analysis

of their genetic interactions.

In this study, we established a method to determine the

frequency of LINE retrotransposition in DT40 cells using

retrotransposition-indicative genes.

2. Materials and methods

2.1. DNA vectors

The vector pEGFP-N1 (Clontech) harbors the gene encoding

enhanced green fluorescence protein (EGFP), and the vector

pGL3-promoter (Promega) harbors the firefly luciferase gene.

The vector pBZ2-5 carries a mneoI-marked wild-type zebrafish

LINE ZfL2-5 copy under the control of the cytomegalovirus

(CMV) promoter (Sugano et al., 2006). The vectors p132.49

and pBB4 are retrotransposition-deficient mutant derivatives of

pBZ2-5, with a point mutation in the RT domain (D689Y) and

deletion of the essential 3 region of the LINE, respectively

(Sugano et al., 2006). The vector p131.11 is a derivative of

pBZ2-5 that was constructed in this study, in which the active

site of the EN domain was disrupted by site-directed

mutagenesis (E72A). The vector pEGFPFLAG-1, used formonitoring transfection efficiencies in the LINE retrotransposi-

tion experiments, was constructed by recloning the EcoRINotI

fragment of pEGFP-N1 (containing the entire EGFP gene) into

the same restriction sites of pFLAG-CMV-5a (SIGMA) to

create a vector that carries the EGFP gene but not the neomycin-

resistance (Neo) gene. The ZfL2-2 vector, designated as pZfL2-

2/mEGFPi, which is marked with the EGFP-based retro-

transposition-indicative gene, was constructed by replacement

of the mneoI region in pBZ2-5 with the EGFP-intron region in

pBS-L1RP-EGFP (Ostertag et al., 2000).

2.2. Cell culture, transfection, and reporter gene expressionmonitoring

Chicken DT40 cells were purchased from RIKEN Biosource

Center (number RCB1464) and were cultured in RPMI medium

1640 (Invitrogen), supplemented with 10% fetal bovine serum

(FBS), 1% chicken serum and 50 M 2-mercaptoethanol in

a humidified atmosphere with 5% CO2 at the temperatures

indicated.

For transfection, exponentially growing DT40 cells

(~7106 cells) were harvested and gently resuspended in

200 l of medium. Cells were then mixed with 15 g of the

vector DNA indicated (or 15 g each of two vectors for

cotransfection experiments), which had been purified using the

Fig. 1. A model for retrotransposition of ZfL2-2 and other LINEs. The original

copy of ZfL2-2 is represented as an open rectangle on the top left, with a single

ORF (gray rectangle) that carries the endonuclease (EN) and reverse

transcriptase (RT) domains. The 3 region of ZfL2-2 essential for retro-

transposition is indicated by a slashed box. Bold arrows indicate the progression

of the LINE retrotransposition steps: transcription, translation, RNP formation,

target DNA cleavage, target-primed reverse transcription (TPRT), and latersteps, which are not yet elucidated.

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QIAfilter Plasmid Mega Kit (Qiagen), and placed in a 3-mm

cuvette. Transfection was carried out by electroporation at

250 V and 960 F on the GENE Pulser (Bio-Rad).

To examine the reporter expression from pEGFP-N1 at

different temperatures, the electroporated cells were diluted into

10 ml of medium and then further diluted by fourfold. Dupli-

cate 10-ml cultures were then incubated at 33 C and 37 C in100-mm dishes. The fraction of EGFP-expressing cells was

determined at each time point using the FACSCalibur Flow

Cytometry System (Becton-Dickinson). The threshold value of

relative fluorescence intensity (RFI) per cell, which discriminates

EGFP-positive and -negative cells, was determined by analyzing

the RFI in cells of an untransfected control culture. The EGFP-

positive region (M1) in Fig. 2B was determined as the region in

which only 0.05% of the control cells were assigned.

To examine the reporter expression from pGL3-promoter,

DT40 cells were cotransfected with pGL3-promoter and pEGFP-

N1. At each time point, cells were sampled, the luciferase activity

was measured by the Steady-Glo Luciferase Assay System(Promega) with the TopCount NXT (PerkinElmer), and EGFP-

expressing cells were counted as described above. For normal-

ization, the relative luminescence units (RLU) of the luciferase

were divided by the number of EGFP-expressing cells in the

culture assayed.

2.3. Retrotransposition assay

DT40 cells were cotransfected with pEGFPFLAG-1 and oneof the LINE expression vectors, pBZ2-2, p132.49, pBB4,

p131.11, or pJM102/L1.3 (Sassaman et al., 1997). After trans-

fection, cells were diluted (see Section 2.2) and incubated at

33 C for 3 days to allow the LINEs to retrotranspose. After this

post-transfection incubation, the numbers of EGFP-positive and

-negative cells were counted by flow cytometry to monitor the

transfection efficiency. For gelation and selection, 5 ml of the

culture (diluted with medium to 1.42.0105 cells/ml, giving a

total of 0.71.0106 cells) in a 100-mm dish was mixed with

an equal volume of agarose-RPMI medium 1640 containing

0.24% agarose, 30% FBS, 3% chicken serum, and 3.2 mg/ml

G418, which had been pre-incubated at 45 C (Adachi et al.,2001). After an 11-day incubation at 37 C to allow the

formation of visible colonies, G418-resistant (G418R) colonies

Fig. 2. Expression of reporter genes in DT40 cells. (A) The fractions of EGFP-expressing cells in transiently transfected cultures. Cells electroporated with pEGFP-N1

were incubated at 33 C (diamonds) and 37 C (triangles) in two independent experiments (closed and open symbols). The percentage of EGFP-expressing cells was

monitored by flow cytometry for 3 days after transfection. (B) Histograms of relative fluorescence intensity (RFI) of EGFP in individual transfected cells. After a 74-

hour incubation at 33 C (upper) or 37 C (lower) after electroporation,the RFIof EGFP in each cell wasmeasuredby flow cytometry. M1 indicates theregion in which

cells were assigned as EGFP-positive based on a negative control experiment (see Section 2.2). (C) Maintenance of luciferaseactivities in DT40 cells transfected with a

transiently introduced vector. Cells electroporated with pEGFP-N1and pGL3-promoter were incubated for 3 days at 33 C (diamonds) or 37 C (triangles), and relative

luminescence units (RLU) of luciferase per EGFP-expressing cell were determined at each time point. Results of two independent experiments are shown (closed and

open symbols). (D)Growth curves of DT40 cells.DT40 cells at a densityof 5 10

5

cells/ml were incubated at 33 C(closedsquares)or 37C (opensquares) for48 h. Ateachtime point, cell densities weremonitored and plotted usinga semi-log scale. The error bars indicate standard deviations of datafrom fourindependent experiments.

118 H. Honda et al. / Gene 395 (2007) 116124


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were counted. Retrotransposition frequency was calculated as

the number of G418R colonies per EGFP-positive cell.

For the EGFP-based retrotransposition-indicative cassette,

DT40 cells were transfected with pZfL2-2/mEGFPi, and the

post-transfection incubation was carried out as described above.

The cells were then observed under a fluorescence microscope

to assess whether any cells expressed active EGFP.

2.4. DNA analysis of the genomes of G418R clones

Four independent G418R colonies were picked and cultured

in new liquid medium at 37 C for 4 to 10 days (until reaching

confluency). Genomic DNAwas then isolated from each culture

using the DNeasy Tissue Kit (Qiagen). PCR was carried out to

examine the structure of the mneoI cassette integrated in these

genomic DNAs with the NeoBglF1 and NeoBamR1 primers

(see Supplementary Table 1). The products were analyzed on a

1% agarose gel. The genomic DNA of untransfected DT40 cells

was used as a negative control.The sequences of the 3 LINE-target junctions were

determined by cassette PCR (Siebert et al., 1995). Genomic

DNA (~1 g) from two of the G418R clones was digested with

EcoRV and SmaI and ligated to a cassette that had been prepared

by annealing of the two oligonucleotides CasL-1 and CasBlu-2.

The ligation products were used as templates for nested PCR. The

first PCR was performed with the mneoI-specific primer F1 and

the cassette-specific primer AP1, and the second PCR was

performed with the ZfL2-2-specific primer F3 and another

cassette-specific primer, AP2. The products of the second PCR

were separated on a 1% agarose gel and analyzed by Southern

hybridization with a 32P-labeled ZfL2-2 probe (P1) to identify

specifically amplified products. We cloned and sequenced two ofthe PCR products. The chromosomal sites of the two insertions

were identified by BLAST search (Altschul et al., 1990) against

the sequenced chicken genome (Hillier et al., 2004). To determine

the sequences of the 5 junctions of these integrants, we designed

primers specific to the upstream (5) regions of the insertions

based on the chicken genome sequenceW1F and W2F for the

integrants on chromosome 24 and 15, respectively. The 5

junction regions were amplified from the genomic DNA by PCR

with NeoBamR1 and the respective chromosome-specific

primers. The amplified fragments were directly sequenced. We

also designed primers specificto the 3 regions of the insertions

W1R and W2R, respectively. Using W1F and W1R (or W2F andW2R), the linearity between the two independently determined

flanking regions in these integrants was confirmed by PCR

analysis.

3. Results

3.1. Reduced temperature improves maintenance of gene

expression from non-replicating vectors in DT40 cells

In human cells, analysis of LINE retrotransposition with the

retrotransposition-indicative cassette mneoI in a well-estab-

lished assay utilizes the episomal replication of the LINE

expression vector (Moran et al., 1996). Such an episomal vector

is, however, not available for chicken DT40 cells at present. We

considered that transient transfection with a non-replicating

vector would be applicable for retrotransposition detection in

DT40 cells as well as in rodent cells (Morrish et al., 2002).

Because LINE retrotransposition requires transcription and

translation (Fig. 1), the level and maintenance of LINE

expression are important factors for efficient retrotransposition.Therefore, we initially examined the effects of incubation

temperature on expression from a transiently introduced DNA

vector. DT40 cells were transfected with the pEGFP-N1

reporter, which encodes EGFP, and the fraction of EGFP-

expressing cells was monitored by FACS analysis for three days

(Fig. 2A). About 20 to 35% of the electroporated cells

expressed EGFP at 26 h after electroporation; the fraction of

total cells in the M1 region approximates the transfection

efficiency (Fig. 2B). EGFP expression was detected even at 74 h

after electroporation, with about 15 to 30% of cells expressing

EGFP (Fig. 2A). The decay slopes of the EGFP-expressing cells

were not affected by the incubation temperatures. However, theRFI of EGFP in the transfected cells was altered by the

incubation temperature (Fig. 2B); most cells incubated at 33 C

had an RFI of 100 to 10,000 or more, whereas the majority of

cells incubated at 37 C had RFIs of 10 to 1000. Therefore,

although the fraction of EGFP-positive cells did not differ, the

expression level in individual cells was significantly diminished

by incubation at 37 C after transfection. We examined this

temperature effect more quantitatively by monitoring luciferase

activity from the transiently introduced reporter pGL3-promoter

vector. At 26 h after electroporation, the luciferase activity was

detected in cultures incubated at both 33 C and 37 C (Fig. 2C).

The RLUs of luciferase per transfected cell were 3 to 4 times

lower when incubated at 37 C compared with 33 C. Moreover,luciferase activity was barely detectable in the culture at 37 C

at 50 and 74 h after electroporation, whereas the culture

incubated at 33C had detectable activity even at 74 h after

electroporation.

The growth rate of DT40 cells was approximately two times

lower at 33 C than at 37 C (Fig. 2D). Because the non-

replicating vectors become diluted through cell division, the

slower growth at 33 C would attenuate the rate of dilution, thus

maintaining a higher level of expression from the vector for a

longer period. Therefore, in the following retrotransposition

studies, we incubated transfected cells at 33 C for 3 days to

allow better expression of the genetically marked LINEs.

3.2. Detection of ZfL2-2 retrotransposition in DT40 cells

ZfL2-2, which is also known as CR1-2_DR (Kapitonov and

Jurka, 2003), is a zebrafish LINE that resembles human LINE-2

(and thus is classified into the L2 clade of LINEs) (Sugano et al.,

2006). ZfL2-2 encodes a single protein containing EN and RT

domains (Fig. 3A). The 3 untranslated region (UTR) of this

element contains a sequence essential for retrotransposition, and

it likely forms a secondary structure when transcribed into

RNA; the UTR ends with a repeated hexanucleotide,

(AAATGT)n (Kapitonov and Jurka, 2003; Kajikawa et al.,

2005; Sugano et al., 2006). We have shown that this zebrafish

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LINE retains retrotransposition activity in human HeLa cells

(Sugano et al., 2006); therefore, we used ZfL2-2 to test if

retrotransposition of genetically marked LINEs is also detect-

able in heterologous chicken DT40 cells.

The LINE expression vector originally used for experiments

with HeLa cells, pBZ2-5 (Sugano et al., 2006), carries an active

genomic copy of ZfL2-2 marked with mneoI (Fig. 3B). In the

mneoI assay, the ZfL2-2 element is placed under the control

of the CMV promoter, and the Neo gene is inserted in the

antisense orientation immediately downstream of the ZfL2-2

ORF. This Neo gene is disrupted by the insertion of an intronsequence in the antisense orientation. Thus, the intron can be

spliced from the LINE transcript but not from the Neo gene

transcript. Transcription, splicing of the marked ZfL2-2 RNA,

reverse transcription of the spliced RNA, and integration of

the resulting cDNA into a chromosomal site restore an intact

Neo gene, allowing phenotypic selection for retrotransposition

(Fig. 3B) (Sugano et al., 2006).

The pBZ2-5 DNA was cotransfected with the EGFP vector

(pEGFPFLAG-1) into DT40 cells, and the cells were incubated

at 33 C for 3 days to allow the LINE to retrotranspose (Fig. 3C).

After this post-transfection incubation, the transfection efficien-

cy was monitored by EGFP expression, and the cells wereseeded into medium in the presence of G418. Because DT40

cells do not adhere to the dish, we used soft agarose plates and

counted the number of G418R colonies that formed. Incubation

for 11 days at 37 C yielded approximately 150 colonies per dish

(Fig. 3D left), suggestive of LINE retrotransposition. The

number of G418R colonies per EGFP-expressing cell, which

was taken as the retrotransposition frequency, was ~1.8103.

Retrotransposition frequency was decreased by 1.5-fold by the

post-transfection incubation at 37 C, presumably reflecting the

lower level of LINE expression.

To confirm that the G418R colonies arose via LINE

retrotransposition, we first examined the effects of LINE

mutations on colony formation. A mutation altering the active

Fig. 3. ZfL2-2 retrotransposition assay in DT40 cells. (A) A schematic of

zebrafish LINE ZfL2-2. The 5 UTR, ORF and 3 UTR are indicated. The EN

and RT domains in the ORF are shown as black rectangles. The slashed box

represents the 3 tail required for ZfL2-2 retrotransposition. (B) Phenotypic

selection of retrotransposition by use of mneoI. The neomycin-resistance gene

(light gray rectangles indicated by N and eo) in the mneoIcassette is flanked

by a promoter (P) and a polyadenylation signal (A) (top row). pCMV, the

cytomegalovirus promoter. SVpA, the SV40 polyadenylation signal. After

transcription and polyadenylation of the LINE RNA (second row), the intron in

mneoI is excised (third row). Reverse transcription of the RNA and cDNA

integration into a new site results in regeneration of the Neo gene (bottom row),

thus conferring G418 resistance to the host cell. (C) Retrotransposition assay in

DT40 cells. The two vectors, pBZ2-5 and pEGFPFLAG-1 (upper left), were co-

introduced into DT40 cells (represented by small open circles in the leftmost

large circle). After post-transfection incubation for 3 days at 33 C, transfection

efficiencies were monitored by determining the fraction of cells expressing

EGFP (represented by small light and dark gray circles in the middle). Among

the transfected cells, only those that had restored the Neo gene by

retrotransposition (represented by small dark gray circles) can form colonies

in a soft agarose plate containing G418 (in the rightmost circle). The panel

shows an image of a colony of G418R DT40 cells formed in the agarose plate.

(D) Retrotransposition-dependent G418R colony formation. Images show 100-

mm dishes with G418R colonies. Cells were transfected with pBZ2-5 (WT),

p132.49 (RTm, an RT mutant), or p131.11 (ENm, an EN mutant). N, the number

of independent experiments; TE, the mean values (standard deviation) for

transfection efficiency; NC, the mean number (standard deviation) of G418R

colonies per 100-mm dish; RF, the mean values (standard deviation) forretrotransposition frequency.

120 H. Honda et al. / Gene 395 (2007) 116124


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site of the RT domain of the protein (D689Y) and deletion of the

3 UTR each disrupt the retrotransposition activity of ZfL2-2 in

HeLa cells (Sugano et al., 2006). When these mutant LINE

vectors were introduced into DT40 cells, no G418R colonies

appeared (Fig. 3D middle and data not shown). In addition,

when a point mutation was introduced into the active site of the

EN domain, again no G418R colonies appeared (Fig. 3D right).These results indicate that the formation of G418R colonies is

completely dependent on the retrotransposition activity of the

LINE that carries mneoI.

In addition, G418R colonies were also formed by transient

transfection of the mneoI-marked pJM102/L1.3 vector, which

carries a human L1 element (Sassaman et al., 1997). The

retrotransposition frequency of human L1 in DT40 cells was

calculated as ~5.9 103, which is ~3 times higher than that of

ZfL2-2. Thus, it is likely that retrotransposition of any type of

active LINE is investigable in DT40 cells.

3.3. Analysis of ZfL2-2 integrants in the genomic DNA of

G418R DT40 cells

To further study the nature of the genetic alterations in the

G418R cells, genomic DNA samples were prepared from four

independent G418R clones. First, we tested for the presence of

the intron-excised derivative of the mneoI cassette in their

genomes. The genomic DNAs prepared were analyzed by PCR

such that both the original (2.1 kb) and intron-excised (1.3 kb)

mneoI could be amplified (Fig. 4A). The intron-less band was

Fig. 4. ZfL2-2 integrants in chromosomal DNA from transfected DT40 cells. (A) Detection of the mneoIcassette DNA in G418RDT40 cells. The DNA fragment of the

mneoIcassette was amplified by PCR with the primers NeoBglF1 and NeoBamR1 (left) and analyzed on an agarose gel (right). pBZ2-5 DNA (lane 1), genomic DNA

of untransfected DT40 cells (lane 2), and genomic DNAs isolated from four independent G418R clones (lanes 36). Lane M, 1-kb plus marker DNAs. (B) The

structures of ZfL2-2 retrotransposition products. Sequences of the integration site pre- and post-integration and schematic representations of the integrants on

chromosomes 24 (Integrant 1) and 15 (Integrant 2) are shown. The sequences of the chicken genome and ZfL2-2 DNA are shown in uppercase and lowercase letters,respectively. The sequence of the ZFL2-2 3 terminal repeat is shown in bold italics. The 5 and 3 microhomologous sequences are underlined.

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indeed amplified from all four genomic DNAs, whereas no

intron-positive band was amplified. These data indicate that the

intron-excised mneoI cassette was indeed inserted and fixed

somewhere in the genomes of the G418R clones and that the

donor LINE vector DNA was lost during the course of

incubation. Second, to study the features of the retrotransposi-

tion products in these cells in detail, we determined thesequences of two retrotransposition sites. The 3 junctions of the

integrants in two of the clones were amplified by cassette PCR

(see Section 2.4). Southern blot analysis of the PCR products

revealed that these clones carried multiple insertions (data not

shown), which also has been observed with the mneoI cassette

in mammalian cells (Wei et al., 2000; Symer et al., 2002).

Sequencing of two of the bands revealed that the LINE was

inserted in chromosomes 24 and 15 of the chicken genome. We

thus designed chromosomal position-specific primers for both

integrations to amplify fragments containing the upstream (5)

region of the target sequence and the inserted copy of ZfL2-2

(see Section 2.4). The integrant on chromosome 24 (Fig. 4B,Integrant 1) carried a 5-truncated ZfL2-2 copy of 4.2 kb

(including the Neo gene) but lacked the intron sequence.

Moreover, the inserted copy ended with the 3 terminal repeat.

These features strongly suggest that this product resulted from

TPRT-initiated retrotransposition rather than another type of

DNA recombination. This ZfL2-2 copy shared microhomolo-

gous sequences with the target sequence at both the 5 and 3

junctions (3 and 5 bp, respectively). Thus, a target sequence of

2 bp, at the longest, was duplicated at each LINE end. The

integrant on chromosome 15 (Fig. 4B, Integrant 2) also

contained a 5-truncated ZfL2-2 copy that ended with the 3

terminal repeat (the length of the insertion was 0.7 kb) and had

microhomologous sequences of 2 and 4 bp at the 5 and 3junctions, respectively. In this instance, a target sequence of at

least 8 bp was truncated. Importantly, all of the features of the

two insertions agreed well with those observed in native ZfL2-2

integrants in the zebrafish genome (Ichiyanagi et al., 2007),

suggesting that retrotransposition in DT40 cells proceeds via

pathways similar to those utilized in nature.

3.4. Detection of retrotransposition using an EGFP-based

retrotransposition-indicative cassette

We also tested if LINE retrotransposition in DT40 cells could

be detected by use of an EGFP-based retrotransposition-indicative cassette (Ostertag et al., 2000). Like mneoI, this

cassette is inactivated by the insertion of an antisense intron and

can only be reactivated by retrotransposition, driving the host

cell to express EGFP. DT40 cells were transfected with pZfL2-

2/mEGFPi, a ZfL2-2 vector containing the EGFP cassette, and

the culture was incubated at 33 C for 3 days. After this

incubation, EGFP-expressing cells were detected by fluores-

cence microscopy; however, the fraction of EGFP-expressing

cells of the total cells transfected was estimated to be only

~ 106 to 105, which was too low to be quantified by flow

cytometry. In addition, when the temperature for the post-

transfection incubation was shifted to 37 C, no EGFP-

expressing cells were observed in ~107 transfectants, consistent

with the results with mneoI that incubation at 33 C yielded

better retrotransposition frequency. Thus, the EGFP cassette,

although detectable at 33 C, is not as appropriate as the mneoI

retrotransposition-indicative cassette for quantitative studies of

LINE retrotransposition.

4. Discussion

In this study, we constructed a new system to detect LINE

retrotransposition in DT40 cells via the mneoI cassette. The

difficulty arising from the absence of an episomal vector that

stably provides LINE transcripts was solved by transient

transfection of a non-replicating vector followed by low-

temperature incubation to maintain a relatively high level of

LINE expression during the period required for a detectable

number of cells to undergo retrotransposition. The second

problem in the previous methodology was the non-adherent

property of DT40 cells, but this was overcome by using the soft

agarose technique (Adachi et al., 2001). Thus, our methodenabled reproducible frequencies of detection of G418R DT40

colonies produced via LINE retrotransposition.

The establishment of this DT40 cell system will allow us to

progress to the next stage of LINE mobility studies. The

involvement of host-encoded proteins in LINE retrotransposi-

tion is very poorly understood, although circumstantial

evidence suggests such involvement (Morrish et al., 2002;

Gilbert et al., 2005; Zingler et al., 2005; Gasior et al., 2006;

Ichiyanagi et al., 2007). DT40 cells are genetically modifiable

by gene targeting, such that mutant derivatives of the gene(s) of

interest can be constructed by knockout (Winding and

Berchtold, 2001; Yamazoe et al., 2004). Therefore, effects of

a series of host mutations on LINE retrotransposition can bestudied by use of isogenic cell lines to identify host-encoded

proteins involved in LINE retrotransposition. Moreover, even

cell lines carrying mutations in multiple genes can be

constructed for DT40 cells; therefore, effects of combinations

of host mutations can be examined by conventional genetic

studies to identify genetic interactions and epistasis groups for

the LINE mobility process. These studies will help to delineate

the LINE retrotransposition pathways, especially the late steps

following the TPRT reaction.

In the course of the study, we also detected retrotransposition

of human L1 in DT40 cells. L1 is distantly related to ZfL2-2 in

the LINE phylogeny (Ohshima and Okada, 2005). L1 and ZfL2-2 show interesting differences in sequence and mobility. First,

L1 carries two ORFs, like many other LINEs, whereas ZfL2-2

has a single ORF. Second, ZfL2-2 retrotransposition requires a

specific sequence in its 3 UTR like many other LINEs (Luan

and Eickbush, 1995; Okada et al., 1997; Kajikawa and Okada,

2002; Osanai et al., 2004; Sugano et al., 2006), whereas L1

retrotransposition does not require such a sequence, but rather

the polyadenosine added at the end of the RNA serves as a

template for reverse transcription (Moran et al., 1996; Cost

et al., 2002; Kulpa and Moran, 2006). Thus, studies of retro-

transposition of these LINEs in wild-type and mutant DT40 cell

lines could reveal general and specific aspects of the modes of

LINE retrotransposition.

122 H. Honda et al. / Gene 395 (2007) 116124


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The retention of LINE retrotransposition activity in heter-

ologous hosts (Eickbush et al., 2000; Kajikawa and Okada,

2002; Sugano et al., 2006, etc) suggests that LINE retro-

transposition involves host proteins highly conserved between

native and experimental model hosts. On the other hand, the

features of the 5 junctions in ZfL2-2 copies that retro-

transposed in human cells have been shown to resemble thoseof L1 integrants in human rather than ZfL2-2 integrants in

zebrafish (Ichiyanagi et al., 2007). This argues in favor of the

idea that the step(s) to join the 5 junction depends on host cell

(or organism) components rather than on the LINEs themselves.

In other words, the retrotransposition intermediates may be

processed by the available host DNA repair system(s). The

relative availability of each of these DNA repair systems may

differ among hosts, resulting in different pathways of retro-

transposition. Recently, a human pre-B cell line, Nalm-6, was

shown to be highly proficient for gene targeting (Adachi et al.,

2006), although not many knockout cell lines have been

constructed. If mutant Nalm-6 cells were compared to mutantDT40 cells in the retrotransposition assay described here, the

effects of equivalent mutations on LINE retrotransposition in

human and chicken could be used to advance our understanding

of the adaptability of LINE mobility pathways as well as host

strategies to adapt to these mobile elements. This information

could improve our understanding of the effects of LINEs on

genomic evolution.

Acknowledgments

We thank Dr. John Moran for generously providing the

plasmid pJM102/L1.3. We thank Drs. Eric Ostertag and Haig

Kazazian for generously providing the plasmid pBS-L1RP-EGFP containing the EGFP retrotransposition-indicative cas-

sette. We thank Mr. Katsumi Yamaguchi for his assistance in the

retrotransposition assay in DT40 cells. Mr. Takayuki Ishino

(Ishiyaku Publishers, Inc.) is acknowledged for his helpful

suggestions on the DT40 experiments. This work was supported

by a Grant-in-Aid to N.O. from the Ministry of Education,

Culture, Sports, Science and Technology of Japan.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.gene.2007.02.017.

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new system for analyzing line retrotransposition in the chicken dt4

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