GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2005) 10, 763-776. doi:10.1111/j.1365-2443.2005.00875.x
© 2005 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, C. W.
Right arrow Articles by Steer, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, C. W.
Right arrow Articles by Steer, C. J.

DNA methylation of Sleeping Beauty with transposition into the mouse genome

Chang Won Park1, Betsy T. Kren1, David A. Largaespada2,3 and Clifford J. Steer1,2,*

1 Department of Medicine,
2 Department of Genetics, Cell Biology and Development, and 3 Arnold and Mabel Beckman Center for Transposon Research, Institute of Human Genetics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The Sleeping Beauty transposon is a recently developed non-viral vector that can mediate insertion of transgenes into the mammalian genome. Foreign DNA elements that are introduced tend to invoke a host-defense mechanism resulting in epigenetic changes, such as DNA methylation, which may induce transcriptional inactivation of mammalian genes. To assess potential epigenetic modifications associated with Sleeping Beauty transposition, we investigated the DNA methylation pattern of transgenes inserted into the mouse genome as well as genomic regions flanking the insertion sites with bisulfite-mediated genomic sequencing. Transgenic mouse lines were created with two different Sleeping Beauty transposons carrying either the Agouti or eGFP transgene. Our results showed that DNA methylation in the keratin-14 promoter and Agouti transgene were negligible. In addition, two different genomic loci flanking the Agouti insertion site exhibited patterns of DNA methylation similar to wild-type mice. In contrast, high levels of DNA methylation were observed in the eGFP transgene and its ROSA26 promoter. These results indicate that transposition via Sleeping Beauty into the mouse genome may result in a significant level of de novo DNA methylation. This may depend on a number of different factors including the cargo DNA sequence, chromosomal context of the insertion site, and/or host genetic background.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Sleeping Beauty (SB) is a recently developed non-viral gene delivery system reconstituted from several inactive Tc1/mariner-type vertebrate transposon elements from a teleost fish genome (Ivics et al. 1997). In its native state, the transposon carried a transposase coding sequence flanked at both ends by terminal inverted and direct repeat sequences (IR/DRs). The obligate SB transposase acts in a cut-and-paste mechanism to insert the IR/DR-flanked transposon into genomic TA dinucleotide target sites. For gene delivery, the original transposase-coding sequence located between the IR/DRs was then replaced with a transgene construct, which can be mobilized by the transposase supplied in either cis (encoded on the same plasmid vector) or trans (codelivery of an additional plasmid or RNA transcript) to the SB transposon. Transposition of transgenes via SB has been demonstrated in cultured mammalian cells (Ivics et al. 1997), mouse ES cells (Luo et al. 1998), liver (Yant et al. 2000), and fertilized eggs (Dupuy et al. 2002). The cross-species activity of the SB transposase was also verified in mouse germ lines (Horie et al. 2001).

Unlike other non-viral gene delivery vectors, SB transposons are designed to insert their transgenes into the mammalian genome, and exhibit long-term, stable expression (Yant et al. 2000). One advantage of SB over vectors derived from viruses is that SB shows no substantial DNA sequence preference for transcribed genomic regions as its target insertion site (Vigdal et al. 2002; Carlson et al. 2003). Biased insertion of viral vectors into transcriptionally active regions of the mammalian genome is a major concern in gene therapy (Scherdin et al. 1990; Schroder et al. 2002; Nakai et al. 2003). While the efficiency of in vivo transfection with SB remains relatively low, improvement in transposition efficiency has been achieved through several modifications (Geurts et al. 2003; Zayed et al. 2004). In addition to its function as a non-viral gene delivery vector, the random nature of SB-transposase mediated transposon insertion has also been used for genomic mutagenesis in mouse (Horie et al. 2001, 2003; Carlson et al. 2003).

Successful gene therapy requires both a high rate of target cell transformation and stable persistent expression of the introduced transgenes. Gene therapy vector systems derived from viruses have shown efficient delivery of transgenes into cells in vivo. However, long-term expression of the transgenes has been associated with significant genomic perturbations, including de novo DNA methylation and transgene inactivation (Jahner et al. 1982; Jahner & Jaenisch 1985). Moloney murine leukemia virus (MoMuLV)-derived retroviral vectors are some of the most efficient vehicles for gene delivery for a variety of cell types. However, DNA methylation of the MoMuLV or its derivatives has been reported in transgenic mouse embryos produced by injection of retroviral DNA into mouse zygote (Jahner et al. 1982), in mouse ES cells (Lei et al. 1996), murine hematopoietic stem cells in vivo (Challita & Kohn 1994), and in murine fibroblast cell lines (Hoeben et al. 1991). In fact, DNA methylation was associated with inactivation of proviruses and transgenes delivered by several different MoMuLV-based retroviral vectors (Jahner et al. 1982; Jahner & Jaenisch 1985; Hoeben et al. 1991; Challita & Kohn 1994). DNA methylation and transgene inactivation have also been observed for mouse stem cell virus (MSCV) LTR-based retroviral vector in tissues of transgenic animals, ES cells, and hematopoietic stem cells (Cherry et al. 2000), murine mammary tumor virus (MMTV) LTR in transgenic animals (Betzl et al. 1996), E2A promoter of adenovirus type2 (Ad2) in transgenic animals (Schumacher et al. 2000), and Rous sarcoma virus in mammalian cells (Searle et al. 1984; Hejnar et al. 1999). In fact, DNA methylation is considered to be a fundamental cellular defense mechanism to prevent the expression of potentially harmful viruses or mobilization of intragenomic transposable elements in mammalian genomes (Yoder et al. 1997).

Thus, in the present study, we investigated whether SB transposition into the mouse genome induced alterations in DNA methylation of the transgenes or genomic DNA flanking the insertion sites. Transgenic mouse lines were created by co-injection of an SB transposon containing an Agouti reporter gene (T/K14A) under control of the keratin-14 (K14) promoter, and mRNA encoding the transposase into one-cell mouse embryos (Dupuy et al. 2002). Other transgenic mouse lines were generated with a different SB transposon (T/MPT-GFP) containing mouse ROSA26 promoter and eGFP transgene (Dupuy et al. 2001; Carlson et al. 2003). The methylation patterns of genomic DNA isolated from the tails of transgenic mouse lines were investigated by bisulfite-mediated genomic sequencing (Grunau et al. 2001). A very low level of DNA methylation in the K14 promoter irrespective of Agouti gene expression was observed while no significant DNA methylation was detected in the CpG-rich region of the Agouti coding sequence. In contrast, SB transposons containing the ROSA26 promoter and eGFP transgene showed a high level of CpG methylation in the mouse genome. These results suggest that SB-mediated gene delivery may have significant influence on DNA methylation of the mammalian genome due to a variety of potential factors.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
DNA methylation of the K14 and Agouti transgene SB transposon

The SB transposon (T/K14A) used to generate the transgenic mice was comprised of the original IR/DR structures of the transposon, a portion of the human keratin-14 promoter (K14) to express the murine Agouti coding sequence (Kucera et al. 1996), and the human growth hormone polyadenylation signal (Fig. 1A) (Dupuy et al. 2002). Full expression of the mouse Agouti transgene turns the coat color of C57BL/6 inbred mouse from black to yellow. The pT/K14A construct was injected into mouse one-cell embryos together with mRNA encoding the SB transposase, resulting in founder mice with genomic T/K14A. The characteristics of the insertions were determined by Southern blot analysis with K14-specific probes as well as ones located in the vector sequences outside the T/K14A in the SB vector plasmid (Fig. 1B). One founder mouse, which had 5 different insertions, was backcrossed with wild-type (C57BL/6), and generated F1 offspring in which the T/K14A insertions were also assessed by Southern analysis. Each different insertion was termed A to E from the largest to the smallest fragment size identified by Southern blot. B- and E-insertions were shown to be transposition events, and segregated independently.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 1  Schematic map, transposition process of the transposon T/K14A, and the T/K14A transgenic animals. (A) Map of T/K14A and schematic diagram of SB-transposase mediated insertion into the genome. The SB transposon T/K14A is composed of the K14 promoter from human keratin-14 gene, the mouse Agouti cDNA as a reporter gene, and the human growth hormone polyadenylation signal (HGpA) between the IR/DRs. The obligate SB transposase binds to the IR/DRs, excises and then inserts the transposon into genomic TA dinucleotide target sites, duplicating the original TA. (B) Generation of T/K14A transgenic mice and determination of their genotype by Southern blot analysis. The pT/K14A construct was co-injected into mouse one-cell embryos with mRNA encoding the required SB transposase. The injected one-cell embryos became founder animals containing 5 different bands by Southern blot utilizing probes specific for the T/K14A transposon, indicating different loci of T/K14A insertions (for details, see Dupuy et al. 2002). Southern blot bands were labeled A to E, from the largest to smallest band size observed. The Southern bands B and E segregated independently in the F1 offspring from the mating of the founder mouse with wild-type animals. Bands C and D cosegregated, implying the same chromosomal origin. The largest band A was presumed to result from a joint fragment from bands C and D. The transgenic progeny harboring a single or multiple copies of the T/K14A transposon were also identified by Southern blot analysis, and DNA isolated from tail clips of these animals was used for the DNA methylation studies.

 
It had been previously demonstrated that the D-insertion was random for pT/K14A (Fig. 1B) (Dupuy et al. 2002), which cosegregated with the C-insertion, suggesting that they both resided in the same chromosome. The A band identified by Southern blot analysis was thought to be a junction fragment formed from the C- and D-insertions. Five of the transgenic offspring with different genotypes and phenotypes were used for the DNA methylation analysis, and included a full-expression yellow mouse (coat and skin including that of tails) with a single, SB-mediated B-insertion of T/K14A; two black mice, each with a single T/K14A E-insertion; an intermediate phenotype mouse with yellow skin (including tail skin) and black hair carrying the cosegregating C- and D-insertions; and a yellow mouse with B-, E-, C-, and D-insertions. The expression of an Agouti transgene in non-Agouti C57BL/6 mice is known to result in varying degrees of yellow coat and/or skin color depending on the expression level (Kucera et al. 1996; Dupuy et al. 2002).

Two CpG-rich regions were identified in the K14 promoter and Agouti transgene by MethPrimer (Li & Dahiya 2002) (Figs 2 and 3). A portion of K14 promoter was selected for bisulfite-mediated genomic sequencing from –413 to –19 with the transcription start site of the Agouti transgene designated as +1 (Fig. 2) (Grunau et al. 2001). This region contains most of the first CpG-rich region. Genomic DNA isolated from the tail clips of the transgenic mice carrying T/K14A was treated with sodium bisulfite, and the –413 to –19 portion of K14 promoter was amplified by PCR. Plasmid clones obtained from the PCR products were sequenced, and the DNA methylation status of each CpG dinucleotide within the K14 promoter region was determined by comparison with original DNA sequence. The DNA methylation pattern of the 11 CpGs in this region was established by sequencing a minimum of 10 clones from the different transgenic mice. A very low level of DNA methylation was detected in this region. The pattern of DNA methylation at the K14 promoter, including the CpG-rich region located just upstream of the Agouti coding sequence was similar across mice with different coat colors (Dupuy et al. 2002). These data suggest that the expression of the Agouti transgene might be regulated by a combination of epigenetic and transcriptional factors. In addition, the chromosomal loci of T/K14A insertion did not noticeably influence the DNA methylation status of the K14 promoter region. In fact, no difference in the DNA methylation pattern was observed between mice with transposed T/K14A (B- or E-insertion) and transgenic animals with random insertion of the transgene (D-insertion).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 2  DNA methylation at the K14 promoter of T/K14A transposon in the transgenic mice. A ~400-bp region (–413 to –19) of the K14 promoter was selected based on computer analysis of the DNA sequence and subjected to bisulfite genomic sequencing. The transcription start site of the Agouti transgene was designated as +1 (rectangular arrow). The selected region spans a portion of the first CpG-rich region (–94 to +11) identified in the T/K14A transposon by computer analysis and contains 11 CpGs. Every line represents a bisulfite PCR clone, in which the methylated and unmethylated CpG dinucleotides are depicted as filled or open circles, respectively. Each separate group of lines represents the bisulfite sequencing data obtained from the individual transgenic mouse whose T/K14A insertion genotype and coat color phenotype related to the expression of the Agouti transgene are indicated at left. Each insertion of the T/K14A was labeled as per the banding pattern shown in Figure 1B. The intermediate phenotype observed for a mouse with only the C- and D-insertions was described as yellow pigmented skin and black hair (see Dupuy et al. 2002).

 
We also applied bisulfite-mediated genomic sequencing analysis to characterize the pattern of DNA methylation at the Agouti coding sequence (Fig. 3). The genomic analysis of several mammalian genes has indicated that DNA methylation can occur even in the transcribed region of genes, tightly regulating their expression (Sengupta et al. 1999; Li et al. 2003). The transcribed sequence within the Agouti transgene is approximately 610 bp in length. The Agouti transcribed sequence is rich in CpG dinucleotides, and contains a large CpG-rich region of ~340 bp identified by MethPrimer computer analysis (Li & Dahiya 2002). The CpG-rich region has 27 CpG dinucleotides in the +235 to +501 region relative to the transcription start site at +1. Genomic DNA isolated from tail clips of T/K14A transgenic mice was used for the bisulfite-mediated sequencing; and the DNA methylation pattern of the CpG-rich region in the Agouti transgene determined by sequencing the bisulfite-PCR clones from each of the five transgenic mice.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 3  DNA methylation at the Agouti transgene of T/K14A inserted in the mouse genome. The major portion (+235 to +501) of a large CpG-rich region (+235 to +580) located by sequence analysis in the transcribed (+1 to +611) region of the Agouti transgene was chosen for bisulfite-mediated genomic sequencing. The selected region contains 27 CpG dinucleotides. The rectangular arrow indicates the transcription start site of Agouti transgene at nucleotide +1. Each line represents a bisulfite PCR clone, in which the methylated CpG dinucleotides are depicted as black circles. Each separate block of lines represents a collection of bisulfite sequencing data obtained from each individual transgenic mouse for which the insertion genotype and coat color phenotype are indicated on the left.

 
The potential for PCR amplification of the endogenous mouse Agouti gene from the bisulfite-treated DNA was excluded due to the presence of an ~2.8 kb intron between bisulfite PCR primers in the endogenous Agouti gene. The preliminary restriction endonuclease digestion required for the efficient denaturation of the genomic DNA in the bisulfite treatment and the short extension time used in the PCR resulted in amplicons of only the size expected from the T/K14A Agouti transgene (data not shown). The 27 CpG dinucleotides in the Agouti transgene were almost devoid of DNA methylation irrespective of the genotypes as well as the phenotypes of the transgenic mice (Fig. 3). Only a few rare methylated CpGs were detected in the 50 amplicons cloned and sequenced from the transgenic animals. Taken together, these data imply that the insertion of the T/K14A SB transposon into a mammalian genome via SB-transposition does not cause a significant level of DNA methylation in at least the promoter and transgene regions within the SB transposon.

DNA methylation of the ROSA26 promoter and eGFP transgene in the mouse genome

To examine the effect of other transgenes on the methylation status of SB, bisulfite-mediated sequencing was applied to transgenic mouse lines created with a T/MPT-GFP SB transposon (Dupuy et al. 2001; Carlson et al. 2003). T/MPT-GFP was constructed with IR/DRs of SB at each end, a splice acceptor from the human HPRT gene (SA), bovine growth hormone polyadenylation signal (pA), the ubiquitous ROSA26 promoter (Kisseberth et al. 1999), a coding sequence for enhanced GFP, and a rabbit ß-globin splice donor (SD) (Fig. 4). This construct was originally designed for poly(A)-trap mutagenesis by disrupting transcription with intronic insertion. T/MPT-GFP was introduced into the FVB/n inbred mouse strain by injecting linearized pT/MPT-GFP plasmid vector together with mRNA encoding the SB10 transposase (Dupuy et al. 2001). One founder mouse line containing a concatamer array of the SB in mouse chromosome 9 was crossed with another transgenic line that ubiquitously expressed the SB10 transposase gene. Doubly transgenic male mice carrying both the T/MPT-GFP concatamer and SB10 transposase gene were bred with female wild-type FVB/n inbred mice, producing progeny that inherited a transposed SB-Tn located at a different chromosomal loci than the original locus of the SB concatamer. Two of the insertions are located in mouse chromosome 8, and one in chromosome 7 (Fig. 4). We investigated the CpG methylation status of three unique single chromosomal insertions of T/MPT-GFP by subjecting genomic DNA isolated from tail clippings to bisulfite sequencing.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 4  CpG methylation with T!=MPT-GFP insertion into the mouse genome. The schematic map of T/MPT-GFP (MPT-GFP SB transposon) shows the typical IR/DRs of SB, the splice acceptor/polyadenylation signal (SA/pA), ROSA26 promoter, eGFP reporter gene, and splice donor (SD) sequence. The ~500-bp region spanning the boundary between the ROSA26 promoter and eGFP coding sequence contains ~350 bp of the 3' end of ROSA26 and ~140 bp of the 5' end of eGFP. This region was examined by bisulfite-mediated genomic sequencing using DNA samples obtained from transgenic mouse lines carrying the inserted T/MPT-GFP in different chromosomes. The putative transcription start site of eGFP is indicated at top by a rectangular arrow. Each line under the map represents bisulfite-PCR subclones with filled (methylated CpG) and open (unmethylated CpG) circles. Bisulfite-sequencing data were grouped together according to the insertion from which each bisulfite subclone was derived (left). Chr., mouse chromosome number.

 
A region of ~500 bp spanning 350 bp of the 3' end of the ROSA26 promoter and about 160 bp of the 5'eGFP-coding sequence was analyzed for CpG methylation. This DNA region contains 57 CpGs, 41 in the ROSA26 promoter and 16 in the eGFP coding sequence. Varying degrees of DNA methylation were observed in the 5' region of the ROSA26 promoter among the different insertions, with significantly more methylation present in the two located in chromosome 8. However, T/MPT-GFP was heavily methylated at the 3' end of the ROSA26 promoter and 5' end of eGFP in all 3 different chromosomal loci with SB insertions. The results clearly indicate that de novo DNA methylation was induced by SB transposition of these DNA elements into the mouse genome. However, the specific conditions for triggering de novo CpG methylation of SB in a mammalian genome are unclear. This observation, combined with those from T/K14A, suggests that DNA methylation associated with SB transposition into the mouse genome is likely to be dependent on many factors, such as DNA sequence of the transgene, mode of transgenesis with SB, or host genetic background.

CpG methylation at the genomic region flanking the E-insertion of T/K14A

No prior sequence information regarding the flanking genomic regions of the C- and randomly integrated D-insertion was available (Dupuy et al. 2002). Therefore, we focused on the independent transposed T/K14A insertions, B and E, for which adjacent mouse genomic DNA sequence had been obtained in the previous study (Dupuy et al. 2002). Two transgenic black mice had a single, SB-mediated E-insertion of T/K14A, and another yellow phenotype mouse also had the E-insertion in addition to B-, C- and D-insertions (Figs 1B and 5). The E-insertion had been localized to mouse chromosome 2, and 54 bp of the genomic flanking sequence had been previously determined (Dupuy et al. 2002). Using this 54-bp DNA sequence, we identified and retrieved from the GENBANK database a unique mouse genomic contig (GENBANK Accession Number NT_078378) containing the 54-bp E-insertion flanking sequence. No CpG-rich region was identified in the 12 kb region flanking E-insertion potentially due to the AT-rich characteristics of the genomic target site preferred by the SB transposon (Carlson et al. 2003).

A 600-bp region exhibiting a relatively high density of CpG dinucleotides was analyzed for potential CpG methylation changes in the flanking region (Fig. 5). This region is ~1 kb downstream to the E-insertion, and only two CpGs are present in the intervening DNA sequence. The flanking genomic region upstream of the E-insertion has only 6 scattered CpG dinucleotides over a 2 kb region. Thus, only CpG methylation status of the 12 CpGs in the 600-bp region ~1 kb 3' of the E-insertion was assessed by bisulfite genomic sequencing. Transgenic mice carrying the E-insertion displayed almost complete methylation of all the CpGs present in this region. The pattern of DNA methylation observed in animals with the E-insertion was not significantly different from that of control mice with another chromosomal locus of T/K14A insertion (Fig. 5, B-insertion). Wild-type inbred animals (C57BL/6) with no SB transposon insertions also exhibited a high level of CpG methylation over this 600-bp region (Fig. 5, WT-1-3). These results indicate that DNA methylation at the chromosomal locus of the E-insertion was not altered by transposition of T/K14A.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 5  CpG methylation at the mouse genomic region flanking the E-insertion of T/K14A. The E-insertion of T/K14A is located in mouse chromosome 2. A 600-bp region located ~1 kb 3' of the E-insertion was subjected to bisulfite genomic sequencing. The vertical lines on the schematic map indicate where the CpG dinucleotides occur in the genomic DNA flanking the E-insertion. The methylation profiles of the 12 CpG dinucleotides in the selected DNA region determined by bisulfite genomic sequencing are depicted as lines in which each circle represents a CpG. The individual lines represent the pattern of methylation observed from the individual clones, in which the CpGs are methylated (•) or unmethylated ({circ}). The coat color phenotypes and types of genomic T/K14A insertion are indicated at left of each block of bisulfite clones collected from an individual transgenic mouse. In addition to data from transgenic mice with the E-insertion, bisulfite sequencing data was obtained from 3 individual C57BL/6 inbred wild-type (WT) mice as endogenous DNA pattern controls. Bisulfite clones from a mouse with the B-insertion of T/K14A were also included as a control for the transposition of an SB transposon within the mouse genome at another locus.

 
Determination of chromosomal locus for the B-insertion of T/K14A and CpG methylation at the adjacent genomic region

We considered that the unperturbed DNA methylation pattern in the adjacent regions to T/K14A transposition could be unique to the genomic locus of the E-insertion. Thus, we examined the B-insertion chromosomal locus carrying a transposed T/K14A. The short 54-bp DNA sequence previously reported for the genomic region flanking the B-insertion was used to search for the mouse genomic contig in the GENBANK database, and resulted in retrieval of two mouse genomic contigs, on chromosome 7 and 13, GENBANK Accession Numbers NT_078548 and NT_078713, respectively. Both contigs contain a DNA sequence > 2.4 kb exhibiting 100% identity prior to divergence (Fig. 6A). The B-insertion is located proximal to the end of the identical sequence, while the 3' portions of the two genomic contigs are different, justifying the chromosomal assignment of 7 or 13. These identical sequences attributed to different mouse chromosomes could be generated either by genomic duplication or by erroneous joining of DNA fragments during the cloning process required for sequencing the mouse genome.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 6  Determination of the chromosomal origin of the B-insertion by PCR. (A) Schematic maps and positions of the primers used in the PCR amplification studies to determine the polarity and chromosomal loci of the T/K14A B-insertion. Two mouse genomic contigs, NT_078548 from mouse chromosome 7 and NT_078713 from mouse chromosome 13, were shown to carry the short genomic sequences identified near the B-insertion of T/K14A. These two genomic contigs possess a > 2.4 kb region of 100% identical sequence, and diverge distal to the site of the B-insertion. Reverse PCR primers, R7 from chromosome 7 and R13 from chromosome 13, were designed according to the unique regions. Forward primers were designed on the basis of two possible orientations of T/K14A relative to the transcription of the Agouti transgene, AF for insertion in the A direction; and BF for the B transcriptional orientation. Four possible primer pairs, AF +R7, AF +R13, BF +R7, and BF +R13, were tested in the subsequent PCR. (B) Among the possible primer pairs described in panel (A), only BF +R7 resulted in the specific amplification of the 1052 bp predicted PCR amplicon only from mice containing the B-insertion (B, at top), indicating that the T/K14A resides in chromosome 7 with the B transcriptional direction of Agouti transgene. Genomic DNA from other transgenic mice with only different insertions (E or C +D) or wild-type mice (WT) was resistant to amplification using these primers. The control PCR indicated that all template DNAs were suitable for PCR amplification, confirming the specificity and authenticity of the B-insertion BF +R7 products. The insertions present in the genomic DNA used as the PCR template are indicated above the lanes, and DNA ladders used to estimate the size of the amplified fragment are shown on the left.

 
In order to determine the correct chromosomal locus of the B-insertion, PCR primers were designed to identify the transposed T/K14A, its authentic chromosomal locus, and the direction of the Agouti transgene transcription (Fig. 6A). The transposition of T/K14A could have occurred in two possible orientations relative to the flanking chromosomal sequences. Therefore, two forward PCR primers, AF and BF, were designed according to the possible A or B orientation of T/K14A, respectively. Two reverse primers, R7 and R13, were also designed on the basis of the two candidate genomic contigs derived from mouse chromosome 7 and chromosome 13, respectively. All four primer pairs, AF +R7, BF +R7, AF +R13, and BF +R13, were used for PCR of genomic template DNA isolated from the various transgenic mice and wild-type controls. A wide range of annealing temperatures was employed to ensure the accuracy of the PCR. Only the BF +R7 primer pair resulted in the successful amplification of a PCR product of the predicted size from the transgenic mice known to carry the B-insertion (Fig. 6B). The other PCR primer pairs resulted in either no detectable amplification or nonspecific amplification even from wild-type mice resulting in amplicons whose size was quite different from the predicted (data not shown). This data clearly indicates that the B-insertion is located in mouse chromosome 7. Therefore, the flanking genomic sequence of the B-insertion was selected from the NT_078548 (chromosome 7) genomic contig, and used in the following study.

The flanking region of the B-insertion demonstrated a very low incidence of CpG dinucleotides (Fig. 7). This observation seems to be closely related to the AT-rich nature of the target site for SB transposition. The 5' flanking sequence of the B-insertion was excluded in the investigation of CpG methylation as the identical > 2.4 kb sequence from chromosome 13 would also be amplified during the PCR using the bisulfite treated DNA templates. Thus, a 650-bp region containing 5 dispersed CpGs located ~630 bp downstream of the B-insertion was subjected to bisulfite sequencing analysis. All 5 CpGs in this region displayed a high level of methylation. No variation in the overall pattern of CpG methylation in the flanking region of the B-insertion was observed in either the transgenic or wild-type mice. Moreover, no significant difference in the DNA methylation status was detected between mice with T/K14A at the B-insertion and control animals. The pattern of CpG methylation in this region was essentially identical in the wild-type (C57BL/6) and transgenic T/K14A mice.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 7  CpG methylation at the mouse genomic region flanking the B-insertion of T/K14A. Due to the possible existence of a putative duplicated region > 2.4 kb in chromosome 13, the 5' genomic flanking region was excluded in the DNA methylation analysis of the B-insertion. The CpGs in the region selected for analysis are depicted as vertical lines on the map. The region selected for the bisulfite genomic sequencing analysis is ~650 bp in length, and located ~635 bp 3' to the B-insertion in mouse chromosome 7. The DNA methylation status at the 5 CpGs within the region of bisulfite genomic sequencing is depicted as blocks with each line representing the sequence data from an individual bisulfite clone. Each grouping of lines represents the information derived from individual transgenic or control wild-type (WT) mice. The other notations are the same as described in earlier figures. Data from the mouse with only the E-insertion were included as a control for the presence of a transposed T/K14A in the genome.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have determined whether insertion of foreign DNA by Sleeping Beauty transposon causes de novo DNA methylation of the cargo transgene and/or the flanking region in the mouse genome. Our data showed that while there was a significant level of DNA methylation associated with transposition of SB carrying a ROSA26 promoter and eGFP transgene, no significant changes were observed when the cargo was a K14 promoter and Agouti transgene. In addition, no detectable effect was seen in the pattern of CpG methylation at genomic regions neighboring the transposed T/K14A SB-transposon compared to wild-type loci without SB insertion.

The SB transposon system has shown significant potential as a non-viral gene delivery vector. Therapeutic transgenes have been delivered and successfully transposed in vivo by hydrodynamic delivery of naked DNA to the liver with codelivery of the SB transposase gene in cis or trans (Yant et al. 2000; Mikkelsen et al. 2003). To ensure persistent and high-level expression of the therapeutic gene product, it is important to identify potential epigenetic modifications, such as DNA methylation, which could lead to silencing of the inserted transgene. In mammals, the transcriptional activity of many genes has been closely linked to DNA methylation or other types of epigenetic changes in histone proteins (Jaenisch & Bird 2003). Introduction of foreign DNA into the mammalian genome has been shown to cause DNA methylation, which represses the activity of the foreign DNA and alters gene expression or proviral activation (Jahner & Jaenisch 1985; Hoeben et al. 1991; Challita & Kohn 1994). Our data provide evidence that the SB transposon system can also induce DNA methylation in the mammalian genome. Specifically, T/MPT-GFP clearly showed high levels of DNA methylation at the ROSA26 promoter and eGFP reporter gene in the mouse genome.

The lack of significant level of DNA methylation observed in T/K14A is in sharp contrast to T/MPT-GFP. A number of differences between T/K14A and T/MPT-GFP transgenic mouse lines may have caused the observed patterns of DNA methylation with SB insertion. The most obvious, of course, is the DNA cargo sequence of T/K14A and T/MPT-GFP, both of which contain DNA sequences foreign to the mouse genome. In addition, T/MPT-GFP also contains splice acceptor as well as donor sites. Currently, it is difficult to speculate on factors that might influence DNA methylation without additional comprehensive studies. The site of insertion is not likely to play a significant role since a similar pattern of DNA methylation was observed at different chromosomal loci carrying T/K14A. The difference in genetic background between the C57BL/6 and FVB/n inbred strains could also contribute to the variation in DNA methylation at the SB site. Additional studies are required to identify the key factors regulating de novo DNA methylation following SB insertion into the mouse genome.

T/MPT-GFP lines were generated from doubly transgenic mice, carrying both the SB10 transposase gene and the T/MPT-GFP SB concatamer. The DNA samples used for analysis came from progeny that had a unique single T/MPT-GFP on chromosomes other than 9, which was the original insertion site. Thus, in each animal analyzed for T/MPT-GFP, SB-mediated transposition must have occurred before or during early spermatogenesis in the doubly transgenic male mice. SB-mediated insertion for T/K14A occurred in either the fertilized egg or at a very early stage of development, depending on the activity of injected mRNA for the SB10 transposase. In addition, the T/K14A insertions segregated independently on the different chromosomes (Dupuy et al. 2002). The modes of transgenesis for T/K14A and T/MPT-GFP could significantly affect the variations observed in the DNA methylation status of SB. In fact, the insertions may have occurred during developmental stages in which overall patterns of DNA methylation in the mammalian genome change (Monk et al. 1987). Very early stages in embryonic development and gametogenesis are known to exhibit extensive erasure and re-establishment of DNA methylation patterns required for germ line or cellular differentiation. Therefore, it would be of interest to assess the immediate epigenetic response of the cell in all stages of gametogenesis and development upon the transposase-mediated insertion of SB into the genome.

Recently, cell-type specific DNA methylation was observed following SB-mediated transposition into a specific mouse chromosomal locus (Yusa et al. 2004). The transposon carrying a hybrid CMV enhancer:ß-actin promoter-driven eGFP transgene located in the mouse Sptlc2 locus exhibited extensive DNA methylation in mouse germ cells, while no apparent CpG methylation of the transposon DNA occurred in mouse ES cells (Yusa et al. 2004). These results support the notion that methylation of the inserted SB can be dramatically different depending on the developmental stage of SB-mediated transposition. Mammalian DNA methylation is known to undergo dramatic changes on a genome-wide scale during gametogenesis and early stages of development (Monk et al. 1987). During reproduction, the original DNA methylation pattern in the one-cell embryo or in the subsequent preimplantation embryo stages immediately after transposition cannot be traced using DNA derived from mouse tail somatic tissues. Moreover, it is difficult to identify the immediate epigenetic response to newly transposed SB, even in cultured proliferating somatic cells. In fact, the original DNA methylation pattern in the transfected cells could be altered in the daughter cells simply through the ensuing rounds of cell replication. However, the epigenetic profile of a stable SB transposon insertion in somatic cells or in non-gametic transgenic animal tissues is more important than the immediate epigenetic response to the SB insertion. In this regard, our present study shows substantial DNA methylation of inserted SB in the transgenic somatic cells.

It is noteworthy that the analysis of CpG methylation status was carried out with DNA isolated from tail tissue of transgenic mice. The K14 promoter derived from the human keratin-14 gene drives expression of the Agouti transgene in the skin of mice (Kucera et al. 1996). In the initial characterization of the animals, it was demonstrated that the tail color of the T/K14A transgenic mice was also modified by the expression of the Agouti transgene (Dupuy et al. 2002). Hence, we could study the DNA methylation status in the mouse tissue where the expression of the transgene could be monitored by the skin and hair color. The low level of DNA methylation in the K14 promoter even in the transgenic animals presenting a black phenotype suggests that the expression of a transgene controlled by K14 may be independent of the level of CpG methylation. To our knowledge, there have been no reports describing the relationship between the activity of the K14 promoter and its DNA methylation status. Thus, the promoter activity of K14 appears to be regulated by mechanisms that are distinct from DNA methylation alone. In this regard, it would be of interest to determine the DNA methylation status of a promoter transposed via SB from a mammalian gene whose expression is clearly dependent on its DNA methylation state, such as BDNF (Chen et al. 2003; Martinowich et al. 2003) or maspin (Futscher et al. 2002).

The relationship between DNA methylation of T/MPT-GFP and its expression is complicated by the mutagenic structure of T/MPT-GFP. The GFP reporter gene in the T/MPT-GFP does not possess a downstream polyadenylation signal. In addition, it contains a splice donor, which must be positioned 5' to a splice acceptor in an endogenous mouse gene for successful transcription and expression. This requires that the GFP coding sequence be in the same orientation for transcription as the endogenous gene for splicing (Dupuy et al. 2001). However, the transcriptional orientation of the T/MPT-GFP 0041 and 0024 insertions was in opposite direction to neighboring endogenous exons, while 0001 was inserted in an intergenic region (Carlson et al. 2003).

In conclusion, our results demonstrate that SB transposition can induce DNA methylation in the mouse genome. This could depend on a number of factors, including the DNA sequence of SB as well as the mode of transgenesis. A more systematic approach will be required to fully analyze potential epigenetic modifications of SB and genomic regions flanking the transposition site. For example, SB constructs carrying DNA sequences from viruses, bacteria, or other vertebrate species might significantly modulate the DNA methylation associated with transposition. Tissue- or chromosomal locus-specific epigenetic modification of the SB transposon may also be key factors. Moreover, further studies are required to evaluate potential epigenetic histone modifications known to affect gene expression and chromatin structure in mammalian DNA. With that information, we can begin to compare the effects of SB transposition on genomic stability and transgene expression with other integrating vectors.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Transgenic animals

Transgenic mouse lines for the T/K14A transposon were created by injection of the SB transposon together with mRNA encoding SB transposase into C57BL/6 mouse one-cell embryos (Dupuy et al. 2002). The founder mouse containing 5 different loci of T/K14A insertions was bred to wild-type C57BL/6 mice, resulting in F1 offspring whose T/K14A insertion sites were determined by Southern blot analysis.

Founder mice for T/MPT-GFP were created by injection of pT/MPT-GFP transposon construct together with SB10 transposase mRNA into the one-cell embryo of FVB/n inbred strain (Dupuy et al. 2001). Doubly transgenic mice were produced by crossing T/MPT-GFP lines to other transgenic FVB/n lines possessing and expressing SB10 transposase ubiquitously. These mice, doubly transgenic for T/MPT-GFP as well as SB10 transposase gene, were subsequently crossed to wild-type FVB/n mice, generating transgenic mice carrying individual SB insertions (mobilized from the T/MPT-GFP concatamer on chromosome 9 by the action of SB10 transposase) on chromosomes different from the original locus on chromosome 9. The transgenic mice used in this study have SB-mediated T/MPT-GFP insertions on chromosomes other than 9.

Bisulfite-mediated genomic sequencing

Genomic DNA isolated from tail clips of transgenic mice was subjected to bisulfite treatment as previously described (Paulin et al. 1998; Grunau et al. 2001) with modifications. Briefly, genomic DNA was digested with restriction endonucleases for efficient denaturation that avoided recognition sites within the regions of insertion. Two µg of genomic DNA purified from the restriction endonuclease digestion was denatured in 0.3 M NaOH for 20 min at 42 °C. A mixture of sodium bisulfite, urea, and hydroquinone was added to the denatured DNA to final concentrations of 5.8 M urea, 3.7 M sodium bisulfite, and 8.8 mM hydroquinone. DNA was incubated in this mixture for 12 h at 55 °C, covered by aluminum foil to prevent light exposure. Subsequently, DNA was purified by QIAEXII resin (Qiagen), treated with 0.3 M NaOH for 20 min at 37 °C, and precipitated by overnight incubation with 3 M ammonium acetate, 1 µg of carrier tRNA (Sigma-Aldrich), and 3 vol of ethanol at –20 °C. Precipitated DNA was dissolved in 30 µL of H2O. Approximately 50 ng of bisulfite-treated DNA was used as the template in subsequent PCR assays. PCR primers were designed on the basis of predicted DNA sequences converted by MethPrimer (Li & Dahiya 2002). Bisulfite PCR primers used for the amplification of the regions investigated for DNA methylation were as follows: K14 promoter forward, 5'-GGAAATTAGGTTTAAGGTGTAGAGGTT-3', and reverse, 5'-TCCATCCTAAAAAACAACACAAATA-3'; Agouti transgene forward, 5'-GTGGTATTGAATAAGAAATTTAAGAAG-3', and reverse, 5'-CTAATTTTAACTTCCACTAAATTTCC-3'; flanking region of the E-insertion forward, 5'-AAGGAATTAAGTAAGGTAGTTAGTTGG-3', and reverse, 5'-TCATACTATACTCCCAAACAAAATCC-3'; flanking region of the B-insertion forward, 5'-GTTAAAGTTAAAAGATTTAGTTGGAG-3', and reverse, 5'-TAACTCTAATATTCTATCTTAACACC-3', T/MPT-GFP forward, 5'-ATGGTGTGTAAAGGTAGTTGAGAAG-3', and reverse, 5'-ACAAATAAACTTCAAAATCAACTTACC-3'. PCR with bisulfite-treated DNA was carried out using a 5 min denaturation step at 94 °C; followed by 35 cycles of 25 s at 94 °C, 20 s at 55 °C, and 35–60 s at 72 °C; with a final extension at 72 °C for 5 min. The extension time at 72 °C for the 35 cycles of amplification was adjusted according to the expected size of the PCR product, and primer-annealing temperature for the flanking region of the B-insertion was 54 °C instead of 55 °C. All PCRs were performed with hot start. After purification with QIAquick Gel Extraction Kit (Qiagen), PCR products were cloned into pGEM®-T Easy vector (Promega). At least 5–15 individual clones containing PCR amplicons from each type of genomic DNA sample were sequenced. We determined the original methylation status of each CpG dinucleotide within a region by comparing the sequences obtained from the cloned PCR products of the bisulfite-treated DNA with the untreated DNA sequences. Only data from bisulfite PCR clones showing complete C-to-T conversion by bisulfite treatment at Cs not located in CpG dinucleotides throughout a region were used.

Determination of the chromosomal locus of the B-insertion of T/K14A by PCR

Forward PCR primers were designed with two probable orientations of the T/K14A: AF, 5'-GACAGGGAATCTTTACTCGG-3'; BF, 5'-AGCCATGACATCATTTTCTGGA-3'. Reverse PCR primers were designed from two potential chromosomal loci for B-insertion: R7 from mouse chromosome 7, 5'-CAGAAGAGTGAATGGAAGCC-3'; and R13 from mouse chromosome 13, 5'-CAGGGACTAAACCACCAATC-3'. PCR amplifications were carried out with four possible combinations of primers: AF +R7, AF +R13, BF +R7, and BF +R13. A wide range of annealing temperatures from 50 to 60 °C were tested for all primer pairs in PCR with genomic DNA templates isolated from the T/K14A transgenic mice. Fifty ng of genomic DNA from various T/K14A transgenic mice as well as from wild-type mice were used for PCR.


   Acknowledgements
 
This research was supported in part by grants from the National Institutes of Health (P01 HL65578 and P01 HL55552), and the Alexander and Margaret Stewart Trust to CJS.


   Footnotes
 
Communicated by: Tetsuya Taga

* Correspondence: E-mail: steer001{at}umn.edu


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Betzl, G., Brem, G. & Weidle, U.H. (1996) Epigenetic modification of transgenes under the control of the mouse mammary tumor virus LTR: tissue-dependent influence on transcription of the transgenes. Biol. Chem. 377, 711–719.[Medline]

Carlson, C.M., Dupuy, A.J., Fritz, S., Roberg-Perez, K.J., Fletcher, C.F. & Largaespada, D.A. (2003) Transposon mutagenesis of the mouse germline. Genetics 165, 243–256.[Abstract/Free Full Text]

Challita, P.M. & Kohn, D.B. (1994) Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc. Natl. Acad. Sci. U S A 91, 2567–2571.[Abstract/Free Full Text]

Chen, W.G., Chang, Q., Lin, Y., et al. (2003) Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 302, 885–889.[Abstract/Free Full Text]

Cherry, S.R., Biniszkiewicz, D., van Parijs, L., Baltimore, D. & Jaenisch, R. (2000) Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol. Cell. Biol. 20, 7419–7426.[Abstract/Free Full Text]

Dupuy, A.J., Clark, K., Carlson, C.M., et al. (2002) Mammalian germ-line transgenesis by transposition. Proc. Natl. Acad. Sci. U S A 99, 4495–4499.[Abstract/Free Full Text]

Dupuy, A.J., Fritz, S. & Largaespada, D.A. (2001) Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88.[CrossRef][Medline]

Futscher, B.W., Oshiro, M.M., Wozniak, R.J., et al. (2002) Role for DNA methylation in the control of cell type specific maspin expression. Nature Genet. 31, 175–179.[CrossRef][Medline]

Geurts, A.M., Yang, Y., Clark, K.J., et al. (2003) Gene transfer into genomes of human cells by the Sleeping Beauty transposon system. Mol. Ther. 8, 108–117.[CrossRef][Medline]

Grunau, C., Clark, S.J. & Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, E65 .

Hejnar, J., Plachy, J., Geryk, J., et al. (1999) Inhibition of the Rous sarcoma virus long terminal repeat-driven transcription by in vitro methylation: different sensitivity in permissive chicken cells versus mammalian cells. Virology 255, 171–181.[CrossRef][Medline]

Hoeben, R.C., Migchielsen, A.A., van der Jagt, R.C., van Ormondt, H. & van der Eb, A.J. (1991) Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position. J. Virol. 65, 904–912.[Abstract/Free Full Text]

Horie, K., Kuroiwa, A., Ikawa, M., et al. (2001) Efficient chromosomal transposition of a Tc1/mariner-like transposon Sleeping Beauty in mice. Proc. Natl. Acad. Sci. U S A 98, 9191–9196.[Abstract/Free Full Text]

Horie, K., Yusa, K., Yae, K., et al. (2003) Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol. Cell. Biol. 23, 9189–9207.[Abstract/Free Full Text]

Ivics, Z., Hackett, P.B., Plasterk, R.H. & Izsvák, Z. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510.[CrossRef][Medline]

Jaenisch, R. & Bird, A. (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genet. 33 (Suppl.), 245–254.

Jahner, D. & Jaenisch, R. (1985) Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity. Nature 315, 594–597.[CrossRef][Medline]

Jahner, D., Stuhlmann, H., Stewart, C.L., et al. (1982) De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature 298, 623–628.[CrossRef][Medline]

Kisseberth, W.C., Brettingen, N.T., Lohse, J.K. & Sandgren, E.P. (1999) Ubiquitous expression of marker transgenes in mice and rats. Dev. Biol. 214, 128–138.[CrossRef][Medline]

Kucera, G.T., Bortner, D.M. & Rosenberg, M.P. (1996) Overexpression of an Agouti cDNA in the skin of transgenic mice recapitulates dominant coat color phenotypes of spontaneous mutants. Dev. Biol. 173, 162–173.[CrossRef][Medline]

Lei, H., Oh, S.P., Okano, M., et al. (1996) De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122, 3195–3205.[Abstract]

Li, L.C. & Dahiya, R. (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 1427–1431.[Abstract/Free Full Text]

Li, H., Myeroff, L., Smiraglia, D., et al. (2003) SLC5A8, a sodium transporter, is a tumor suppressor gene silenced by methylation in human colon aberrant crypt foci and cancers. Proc. Natl. Acad. Sci. U S A 100, 8412–8417.[Abstract/Free Full Text]

Luo, G., Ivics, Z., Izsvák, Z. & Bradley, , A. (1998) Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells. Proc. Natl. Acad. Sci. U S A 95, 10769–10773.[Abstract/Free Full Text]

Martinowich, K., Hattori, D., Wu, H., et al. (2003) DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science 302, 890–893.[Abstract/Free Full Text]

Mikkelsen, J.G., Yant, S.R., Meuse, L., Huang, Z., Xu, H. & Kay, M.A. (2003) Helper-independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo. Mol. Ther. 8, 654–665.[CrossRef][Medline]

Monk, M., Boubelik, M. & Lehnert, S. (1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382.[Abstract]

Nakai, H., Montini, E., Fuess, S., Storm, T.A., Grompe, M. & Kay, M.A. (2003) AAV serotype 2 vectors preferentially integrate into active genes in mice. Nature Genet. 34, 297–302.[CrossRef][Medline]

Paulin, R., Grigg, G.W., Davey, M.W. & Piper, A.A. (1998) Urea improves efficiency of bisulphite-mediated sequencing of 5'-methylcytosine in genomic DNA. Nucleic Acids Res. 26, 5009–5010.[Abstract/Free Full Text]

Scherdin, U., Rhodes, K. & Breindl, M. (1990) Transcriptionally active genome regions are preferred targets for retrovirus integration. J. Virol. 64, 907–912.[Abstract/Free Full Text]

Schroder, A.R., Shinn, P., Chen, H., Berry, C., Ecker, J.R. & Bushman, F. (2002) HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529.[CrossRef][Medline]

Schumacher, A., Koetsier, P.A., Hertz, J. & Doerfler, W. (2000) Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice. J. Biol. Chem. 275, 37915–37921.[Abstract/Free Full Text]

Searle, S., Gillespie, D.A., Chiswell, D.J. & Wyke, J.A. (1984) Analysis of the variations in proviral cytosine methylation that accompany transformation and morphological reversion in a line of Rous sarcoma virus-infected Rat-1 cells. Nucleic Acids Res. 12, 5193–5210.[Abstract/Free Full Text]

Sengupta, P.K., Ehrlich, M. & Smith, B.D. (1999) A methylation-responsive MDBP/RFX site is in the first exon of the collagen {alpha}2 (I) promoter. J. Biol. Chem. 274, 36649–36655.[Abstract/Free Full Text]

Vigdal, T.J., Kaufman, C.D., Izsvak, Z., Voytas, D.F. & Ivics, Z. (2002) Common physical properties of DNA affecting target site selection of Sleeping Beauty and other Tc1/mariner transposable elements. J. Mol. Biol. 323, 441–452.[CrossRef][Medline]

Yant, S.R., Meuse, L., Chiu, W., Ivics, Z., Izsvá, k, , Z. & Kay, M.A. (2000) Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nature Genet. 25, 35–41.[CrossRef][Medline]

Yoder, J.A., Walsh, C.P. & Bestor, T.H. (1997) Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 13, 335–340.[CrossRef][Medline]

Yusa, K., Takeda, J. & Horie, K. (2004) Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation. Mol. Cell. Biol. 24, 4004–4018.[Abstract/Free Full Text]

Zayed, H., Izsvá, k, Z., Walisko, O. & Ivics, Z. (2004) Development of hyperactive Sleeping Beauty transposon vectors by mutational analysis. Mol. Ther. 9, 292–304.[Medline]

Received: 12 March 2005
Accepted: 18 April 2005




This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
W. Wang, C. Lin, D. Lu, Z. Ning, T. Cox, D. Melvin, X. Wang, A. Bradley, and P. Liu
Chromosomal transposition of PiggyBac in mouse embryonic stem cells
PNAS, July 8, 2008; 105(27): 9290 - 9295.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
B. S. Garrison, S. R. Yant, J. G. Mikkelsen, and M. A. Kay
Postintegrative Gene Silencing within the Sleeping Beauty Transposition System
Mol. Cell. Biol., December 15, 2007; 27(24): 8824 - 8833.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, C. W.
Right arrow Articles by Steer, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, C. W.
Right arrow Articles by Steer, C. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS