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Developmental Stage-Specific Regulation of TCR--Chain Gene Assembly by Intrinsic Features of the TEA Promoter¹

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

	Abstract

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The TCR - and -chain genes lie in a single complex locus, the TCR/ locus. TCR-chain genes are assembled in CD4^–CD8^– (double negative (DN)) thymocytes and TCR-chain genes are assembled in CD4⁺CD8⁺ (double positive) thymocytes due, in part, to the developmental stage-specific activities of the TCR and TCR enhancers (E and E), respectively. E functions with TCR promoters to mediate TCR-chain gene assembly in DN thymocytes. However, E is unable to function with TCR promoters such as the TEA promoter to drive TCR-chain gene assembly in these cells. This is important, because the premature assembly of TCR-chain genes in DN thymocytes would disrupt and T cell development. The basis for TEA inactivity in DN thymocytes is unclear, because E can activate the V5 gene segment promoter that lies only 4 kb upstream of TEA promoter. In this study, we use gene targeting to construct a modified TCR/ locus (TCR/^5T) in which the TEA promoter lies in the same location as the V5 gene segment on the wild-type TCR/ allele. Remarkably, the TEA promoter on this allele exhibits normal developmental stage-specific regulation, being active in double positive thymocytes but not in DN thymocytes as is the case with the V5 promoter. Thus, the inactivity of the TEA promoter in DN thymocytes is due primarily to intrinsic developmental stage-specific features of the promoter itself and not to its location relative to other cis-acting elements in the locus, such as E.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
T cell progenitors pass through a series of developmental stages in the thymus where they must satisfy specific requirements centered around the assembly and expression of TCR genes if they are to be released into the periphery as mature or T cells (1). Assembly of the TCR-, -, and -chain genes is initiated in double negative (DN)³ thymocytes (2, 3, 4). Expression of a TCR leads to progression along the T cell lineage pathway. Generation of a productive TCR-chain gene and expression of a TCR-chain, as a pre-TCR, promote commitment to the T cell lineage, cellular expansion, and transition to the double positive (DP) stage of thymocyte development (5). Pre-TCR signals also lead to the initiation of TCR-chain gene assembly, which occurs primarily in DP thymocytes, as well as the cessation of further TCR-chain gene rearrangements through the process of allelic exclusion (2, 3, 5, 6, 7).

The assembly of different TCR genes is precisely regulated within the context of thymocyte development. This regulation is mediated, in part, by modulation of the accessibility of gene segments to the RAG-1 and -2 proteins through the activity of cis-acting elements, such as promoters and enhancers, that regulate transcription (8). The regulation of the TCR- and -chain genes poses perhaps the greatest regulatory challenge, as these genes lie in a single complex locus, the TCR/ locus (2, 9). The TCR/ locus spans 1.6 Mb with the V and V gene segments clustered over 1.5 Mb in the 5' region of the locus (Fig. 1) (9, 10). Some V gene segments are used primarily in TCR-chain gene assembly (V gene segments), and others primarily in TCR-chain gene assembly (V gene segments). However, most V gene segments are used in both TCR- and TCR-chain gene assembly (V/ gene segments) (10). The V gene segment cluster is followed by two D (D1 and D2) gene segments, two J (J1 and J2) gene segments, and the TCR constant region gene (C) (Fig. 1). A single V gene segment, V5, lies 3' of C and rearranges by inversion (Fig. 1). A cluster of 61 J gene segments and the TCR constant region gene (C) lie in the most 3' region of the locus (Fig. 1).

View larger version (5K): [in this window] [in a new window]   FIGURE 1. Schematic of the TCR/ locus. Shown are the V/V segments (filled rectangle), J segments (gray rectangle), E (gray oval), E (filled oval), C exons (open rectangle), V5 RSS (open triangle), TEA promoter (open diamond), TEA exon (hatched rectangle), and the J2/J61 RSS (black triangle). Arrows indicate the direction of transcription. The schematic is drawn to scale.

TCR gene assembly does not occur in DN thymocytes due, at a minimum, to the inactivity of the TEA promoter and other J gene segment promoters in these cells (13). This may result from the inactivity of E in DN thymocytes (11, 14). However, E is active in DN thymocytes and promotes the accessibility of TCR gene segments, including V5 (see Results), that lie immediately upstream of the TEA promoter (2, 15). How is it that E is capable of activating the V5 promoter, which lies 10 kb downstream, but not the TEA promoter, which lies 14 kb downstream (Fig. 1)? Deletion of a 2-kb region between V5 and the TEA promoter known as BEAD-1, which exhibits insulator activity in vitro, did not lead to TEA promoter activation in DN thymocytes (16). It is thus possible that other sequences between V5 and the TEA promoter, such as the V5 promoter, prevent E from functioning with the TEA promoter. In addition, because E may function over a shorter distance than E it is possible that E may function over the 10-kb distance to V5 but not the 14-kb distance to TEA (17). Lastly, it is possible that intrinsic features of the TEA promoter prohibit its activation in DN thymocytes.

To distinguish between these possibilities, we used a gene-targeting approach to generate mice with a modified TCR/ locus (TCR/^5T), in which a 4-kb region encompassing the V5 gene segment and the immediate downstream region to the 5' border of the TEA promoter has been deleted. Thus, the TEA promoter on the TCR/^5T allele lies in the same location as the V5 gene segment on the wild-type TCR/ allele. The TEA promoter on the TCR/^5T allele exhibits normal activity in DP thymocytes. However, analyses of TCR/^5T/5T DN thymocytes failed to reveal a significant increase in TEA-driven J gene segment transcription or accessibility. These findings demonstrate that the inactivity of the TEA promoter in DN thymocytes is not due to its relative position in the locus with respect to other cis-acting elements such as E. Rather, our findings suggest that intrinsic features of the TEA promoter limit its activity in DN thymocytes and, as such, have important implications for the developmental stage-specific regulation of TCR gene assembly.

	Materials and Methods

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Mice

All mice were bred and maintained under specific pathogen-free conditions at the Washington University School of Medicine (St. Louis, MO) and were handled in accordance to the guidelines set forth by the Division of Comparative Medicine of Washington University.

Generation of embryonic stem cells with the TCR/^5T allele

The 5' homology arm of the pV5^TC targeting vector was generated as PCR fragment corresponding to nt 8574–14306 of the GenBank accession no. M64239 sequence. The 5' end of this fragment is in exon 1 of the C gene segment and the 3' end is in the spacer of V5 gene segment recombination signal sequences (RSS). The 3' homology arm is a BglII-NsiI DNA fragment corresponding to nt 18292–25232 of the GenBank accession no. M64239 sequence. The 5' end of this fragment is immediately upstream of the 5' border of the TEA promoter. The homology arms were cloned into the pLNTK vector (12). Embryonic stem cell targeting and Cre-loxP mediated deletion was conducted as previously described (12).

Southern and Northern blot analyses

Southern and Northern analyses were performed as described (12). The 5' (probe 5), V5 (probe 7), C (probe 6), TEA (probe 8), C, and GAPDH probes have been described (12, 18). The 3' probe is a PCR fragment corresponding to nt 26554–27388 of the GenBank accession no. M64239 sequence.

PCR, RT-PCR, J utilization index calculation, and ligation-mediated PCR (LMPCR)

VDJ rearrangements were assayed by PCR using the oligonucleotides 5'-TTTTGGTATCGCAAAAGGCC-3' and 5'-CCCTGCTCCTATGGAGGAGC-3'. Standard PCR buffer conditions with 1 mM MgCl₂ were used. Cycling conditions were 94°C for 5 min followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. This was followed by a final 7 min of incubation at 72°C. PCR products were hybridized to the oligonucleotide probe 5'-TGTGAAGCACAGCAAGGCCA-3' as described previously (19). The IL-2 gene PCR was described previously (20).

RT-PCR for analysis of J gene segment utilization has been described previously (21). The J gene segment utilization index was calculated as index = (J^5T/C^5T)/(J⁺/C⁺), where J^5T and J⁺ are the intensities of the Southern blot bands from the hybridization of J-specific oligonucleotides to V-C RT-PCR products from TCR/^5T/5T and wild-type splenocyte cDNA, respectively. C^5T and C⁺ are the intensities of the Southern blot bands from the hybridization of a C-specific oligonucleotide to the same RT-PCR products and serve as the loading control. LMPCR and Southern analyses were conducted with primer A (5'-CCAAGATTCCTGGGACAACC-3'), primer B (5'-GCGATGGGACTGTGACTGAC-3'), primer BW1H, and the oligonucleotide probe P (5'-TCCAAAAGAGGAAAGGAAGGCAGTC-3') as previously described (19).

Flow cytometric analyses and cell sorting

To obtain DN CD25⁺ cells, total thymocytes were enriched for DN cells by complement-mediated lysis of CD4⁺ and CD8⁺ cells as described (19). The resulting cells were stained with FITC-conjugated anti-mouse CD25 (BD Pharmingen) followed by sorting for CD25⁺ cells with FACSDiva (Becton Dickinson) to >95% purity.

Generation of T cell hybridomas

Two independent panels of T cell hybridomas were generated from TCR/^+B6/5T mice as described previously (12).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Optimal V5 transcription and accessibility in DN thymocytes requires E

To determine whether E is required for V5 gene segment transcription and accessibility in DN thymocytes, we conducted Northern blot analyses on RNA from RAG-2-deficient DN thymocytes that were homozygous for the wild-type TCR/ locus (R2^–/–:E^+/+) or a TCR/ locus in which E had been deleted (R2^–/–:E^/) (Fig. 2A). R2^–/–:E^+/+ DN thymocytes have a robust level of V5-hybridizing germline transcripts (Fig. 2A). In contrast, the level of V5 germline transcripts were dramatically reduced in R2^–/–:E^/ DN thymocytes, demonstrating that E activity is required for the optimal level of V5 gene segment transcription in these cells (Fig. 2A). That E is required for V5 rearrangement is evidenced by the 10-fold reduction in complete V5DJ1 rearrangements in E^/ thymocytes as compared with wild-type thymocytes (Fig. 2B). In agreement with previous reports, TEA-hybridizing transcripts were not observed in DN thymocytes (Fig. 2C) (13). Together, these findings demonstrate that in DN thymocytes E functions to promote V5 gene segment transcription and accessibility but it is unable to activate the TEA promoter, which lies just 4 kb downstream of V5.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. E is required for V5 germline transcripts and efficient VDJ rearrangement. A, Northern blot analyses of V5 germline transcripts in RAG-2–/–:E/ (R2–/–:E/) and RAG-2–/–:E+/+ (R2–/–:E+/+) thymocytes and RAG-2–/– splenocytes (Spl). Northern blot hybridization was conducted using the V5 or GAPDH (G) probes. The positions of 28S and 18S RNA are indicated. B, VDJ rearrangements in E/ or wild-type (E+/+) thymocytes were assayed by PCR as described in Materials and Methods. Shown are 4-fold serial dilutions of template DNA. PCR products corresponding to VD or VDJ rearrangements are indicated. IL-2 PCR as a DNA loading control is also shown. C, Northern blot analyses of germline TEA transcripts in RAG-2–/– (R2–/–) DN or RAG-2–/–:DO11.10 TCR transgenic (R2–/–:) DP thymocytes.

Generation of TCR/^5T/5T mice

To determine why E is unable to activate the TEA promoter in DN thymocytes, we generated the TCR/^5T allele through gene targeting of embryonic stem cells (Fig. 3). Initially the TCR/^5NT allele was generated by replacing the 4-kb region upstream of the TEA promoter that includes the V5 gene segment with the neomycin resistance gene flanked by loxP sites (Fig. 3, A and B). The TCR/^5T allele was then generated from the TCR/^5NT allele through Cre-loxP mediated deletion of the neomycin resistance gene (Fig. 3). On the TCR/^5T allele the TEA promoter lies at the same distance from E as the V5 gene segment on the wild-type TCR/ allele (Fig. 3A). Furthermore, any sequences between V5 and the TEA promoter that could inhibit E interactions with the TEA promoter, including the V5 promoter, have been deleted on the TCR/^5T allele.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Generation of the TCR/5T allele. A, Schematic of the wild-type allele (TCR/+), the targeting construct (pV5TC), the targeted allele retaining the neomycin-resistant gene (TCR/5NT), and the TCR/5NT allele after deletion of the neomycin-resistant gene (TCR/5T). The C exons (gray rectangle), V5 gene segment, TEA exon (open rectangle), J segments (small gray rectangle), neomycin-resistant gene (NEOr; open box), TEA promoter (open diamond) and loxP sites (open triangles) are shown. The BamHI (B) and BglII (G) sites are indicated. Arrows indicate the direction of the V5 and TEA transcripts. The locations of 5' probes, 3' probes, and the TEA probe (thick line) are shown. The schematic is drawn to scale. B and C, Southern blot analyses of ES cells with TCR/+ (+), TCR/5NT (5NT), and TCR/5T (5T) alleles. Genomic DNA was digested with BamHI and the probes used for hybridization are indicated. See A for schematic.

5T/5T

The TEA promoter on the TCR/^5T allele is active in DP thymocytes

To determine whether the TEA promoter on the TCR/^5T allele is regulated normally in DP thymocytes, we generated RAG-2-deficient mice expressing the DO11 TCR transgene that were homozygous for the wild-type TCR/ allele (R2^–/–::TCR/^+/+) or the TCR/^5T allele (R2^–/–::TCR/^5T/5T) (Fig. 4A). Northern blot analysis revealed similar levels of germline TEA-hybridizing transcripts in R2^–/–::TCR/^+/+ and R2^–/–::TCR/^5T/5T DP thymocytes (Fig. 4A). Analysis of TCR mRNA from TCR/^5T/5T and TCR/^+/+ T cells revealed that similar fractions of VJ rearrangements use the J58 and J57 gene segments, which are located 5' in the J gene segment cluster and are dependent on TEA promoter activity for accessibility (Fig. 4B) (22). Together, these data demonstrate that the TEA promoter is intact and regulates transcription and accessibility normally on the TCR/^5T allele in DP thymocytes.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. TEA promoter activity on the TCR/5T allele in DP thymocyte. A, Northern blot analyses of TEA transcripts in R2–/–::TCR/+/+ and R2–/–::TCR/5T/5T thymocytes using the TEA and GAPDH (G) probes for hybridization. The position of 28S and 18S RNA are shown. B, J utilization. Amplified TCR cDNA from TCR/5T/5T or wild-type (TCR/+/+) splenic T cells was hybridized to different J gene segment-specific oligonucleotides and a C-specific oligonucleotide as described in Materials and Methods. The index number that reflects the relative J oligonucleotide hybridization in the TCR/5T/5T and TCR/+/+ TCR cDNAs is indicated and was calculated as described in the Materials and Methods section.

Minimal TEA promoter activity on the TCR/^5T allele in DN thymocytes

Transcription from the TEA promoter in DN thymocytes was assayed by the Northern blotting of RNA isolated from RAG-2^–/–:TCR/^+/+ (R2^–/–:TCR/^+/+) and RAG-2^–/–:TCR/^5T/5T (R2^–/–:TCR/^5T/5T) thymocytes (Fig. 5). As shown previously, no TEA- or C-hybridizing transcripts were detectable in R2^–/–:TCR/^+/+ (Fig. 5). Although there were slightly more TEA-hybridizing transcripts in DN thymocyte RNA from R2^–/–:TCR/^5T/5T as compared with R2^–/–:TCR/^+/+ mice, the level was significantly lower than what was observed in R2^–/–::TCR/^5T/5T DP thymocytes where the TEA promoter is active (Fig. 5). Furthermore, although TEA transcripts are frequently spliced to C, C hybridizing transcripts were not detected in R2^–/–:TCR/^5T/5T DN thymocytes (Fig. 5). Germline TCR transcripts from the TCR/⁺ and TCR/^5T alleles were found at similar levels in DN thymocytes (Fig. 5). Together, these data demonstrate that although E is functional and promotes normal level of germline TCR transcripts on the TCR/^5T allele in DN thymocytes (Fig. 5), it is unable to promote a significant level of TEA transcripts in these cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Minimal TEA promoter on the TCR/5T allele in DN thymocytes. Northern blot analysis was conducted as described in the Fig. 2 legend using RAG-2–/– splenocyte (Spl) RNA or RNA from thymocytes as described in Results. RNA was subjected to hybridization with the TEA exon (TEA), C, C, or GAPDH (G) probes, The positions of 28S and 18S RNA are shown.

J gene segment accessibility on the TCR/^5T allele in DN thymocytes

To determine whether J gene segments are accessible for RAG-mediated cleavage, we assayed for J61 signal ends (SEs) in thymocytes at different stages of development by LMPCR. To this end, thymocytes from mice heterozygous for the wild-type B6 and 129 TCR/ alleles (TCR/^+B6/+129) or those hemizygous for the wild-type B6 TCR/ allele and the TCR/^5T allele (TCR/^+B6/5T) were assayed. The TCR/^5T allele was generated through targeting of the 129 TCR/ allele. LMPCR products generated by TCR/⁺¹²⁹ or TCR/^5T J61 SEs can thus be distinguished from TCR/^+B6 J61 SEs by a DdeI restriction site polymorphism (Fig. 6A).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. RAG-mediated cleavage at J61 on the TCR/5T and TCR/+ alleles in DN and DP thymocytes. A, Schematic of LMPCR for J61 SEs. J61 RSS (open triangle), the BW linker (double line), and oligonucleotides used for PCR (arrow) and the oligonucleotide probe (P) are shown. The DdeI site and the expected PCR product sizes following DdeI digestion of products from the 129 (+129), B6 (+B6) and TCR/5T (5T) alleles are also indicated. B, LMPCR for J61 SEs in DNA from total thymocytes or CD25+ DN thymocytes from TCR/+B6/+129 (+B6/+129) or TCR/+B6/5T (+B6/5T) mice as described in Materials and Methods. Serial 4-fold dilutions are shown. A no-ligase added (–Lig) control and IL-2 gene PCR for DNA loading are also shown. C and D, LMPCR products from B were run undigested (–) or after digestion with DdeI (+). The LMPCR products corresponding to J61 SEs from the +129/5T alleles or the +B6 allele after digestion with DdeI are indicated.

+B6/5T

5T

5T

To determine whether the TEA promoter on the TCR/^5T allele can promote J gene segment accessibility in DN thymocytes, CD25⁺ DN thymocytes were purified from TCR/^+B6/5T and TCR/^+B6/+129 mice by flow cytometric cell sorting. CD25⁺ DN thymocytes from TCR/^+B6/5T and TCR/^+B6/+129 mice had similar levels of J61 SEs, which were considerably lower than the levels observed in DP thymocytes (Fig. 6B). Furthermore, digestion of the LMPCR products with DdeI revealed that there were similar levels of J61 SEs from the TCR/^+B6 allele and TCR/^5T allele in TCR/^+B6/5T DN thymocytes (Fig. 6D). Together, these findings demonstrate that there is a significantly lower level of RAG-mediated cleavage at the J61 gene segment on the TCR/^5T allele in DN thymocytes as compared with DP thymocytes. Furthermore, the level of J61 gene segment cleavage on the TCR/^5T allele is similar to that observed for the wild-type TCR/ allele in DN thymocytes. Thus, the TEA promoter does not promote increased J gene segment accessibility on the TCR/^5T allele in DN thymocytes.

V to J rearrangement is not initiated on the TCR/^5T allele in T cells

E may only function with the TEA promoter on the TCR/^5T allele in DN thymocytes that have committed to the T cell lineage and promote V-J rearrangements in these cells. To investigate this possibility, we generated a panel of 81 T cell hybridomas from splenic TCR/^+B6/5T T cells. That these T cell hybridomas were all derived from T cells was evidenced by flow cytometric analyses demonstrating cell surface expression of TCR- but not TCR-chains (data not shown).

All V to J rearrangements occur by deletion and result in deletion of the TEA exon from the TCR/^+B6 or TCR/^5T allele in TCR/^+B6/5T T cells. Genomic DNA isolated from the TCR/^+B6/5T T cell hybridomas was digested with BglII and subjected to Southern blot analyses using the TEA exon probe (Fig. 7). A BglII polymorphism, generated by the targeting, permits the distinction of TEA hybridization on the TCR/^+B6 or TCR/^5T alleles in TCR/^+B6/5T T cells (Fig. 3A). These analyses revealed the retention of TEA hybridization on both the TCR/^+B6 and TCR/^5T alleles in all 81 of the TCR/^+B6/5T T cell hybridomas (Fig. 7 and data not shown). That these hybridomas represented a diverse population of T cells was evidenced by hybridization with a probe 3' of the J1 gene segment, which revealed many different size-hybridizing fragments that represent heterogeneous TCR-chain gene rearrangements in the TCR/^+B6/5T T cell hybridomas (Fig. 7). Thus, E is not capable of promoting V to J gene segment rearrangement on the TCR/^5T allele in T cell precursors.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. V-J rearrangements are not found on the TCR/5T allele in T cells. Southern blot analyses of genomic DNA from individual TCR/+B65T T cell hybridomas (lanes 1–5), as well as control kidney DNA from B6 (+/+) or TCR/5T/5T (5T/5T) mice digested with BglII and probed with the TEA or 3' J1 probes. The positions of germline fragments for the B6 (+) or TCR/5T allele (5T) are indicated. See Fig. 3A for a schematic of Southern analyses.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The TEA promoter is factor-loaded in DN thymocytes but is unable to function in these cells (23). It is possible that transcription factors bound to the TEA promoter in DN thymocytes are repressive in nature. However, it is also possible that some additional trans-acting factors are required for TEA activity but are only expressed in DP thymocytes. In this regard, it is notable that the expression of Ets family transcription factors, which are predicted to bind to the TEA promoter, are up-regulated as cells transit from the DN to the DP stage of thymocyte development (24). It is also possible that the TEA promoter has a 5' region with insulator activity.

Because many V gene segments in the TCR/ locus are accessible and participate in TCR gene assembly in DN thymocytes, it is likely that V to J rearrangements are prevented in these cells primarily due to the inaccessibility of the J gene segments. Our findings suggest that this inaccessibility is due, in part, to intrinsic features of the TEA promoter that prohibit it from being activated by cis-acting elements, such as E, that function to promote TCR-chain gene rearrangements in DN thymocytes.

Activation of V to J rearrangements in DN thymocytes could have a detrimental effect on both and T cell development and lineage commitment. All V to J rearrangements occur by deletion, leading to the excision of the TCR-chain genes from the chromosome and, thus, limit the ability of DN thymocytes to generate a TCR (25). Expression of an TCR or a pre-TCR by DN thymocytes leads to signals that induce transition to the DP stage of thymocyte development (5). However, the pre-TCR is more efficient at mediating this transition than the TCR (26, 27). The TCR-chain can compete successfully with the pre-T protein for dimerization with the TCR-chain (28). Thus, a productive VJ rearrangement and the expression of a TCR-chain in DN thymocytes would favor formation of an TCR instead of pre-TCR and, as a result, promote a less efficient transition to the DP stage of thymocyte development.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ B.P.S. is supported by National Institutes of Health Grants AI47829 and AI49934 and American Cancer Society Grant RSG-05-070-01-LIB.

² Address correspondence and reprint requests to Dr. Barry P. Sleckman, Department of Pathology and Immunology, 660 South Euclid Avenue, Campus Box 8118, Washington University School of Medicine, St. Louis, MO 63110. E-mail address: Sleckman{at}immunology.wustl.edu

³ Abbreviations used in this paper: DN, double negative; DP, double positive; LMPCR, ligation-mediated PCR; RSS, recombination signal sequence; SE, signal end.

Received for publication March 20, 2007. Accepted for publication April 29, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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