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TCR receptors in other locations show much more diversity. Many intraepithelial T cells resident in the intestinal epithelium use V5 associated with various V chains, but these chains, unlike the invariant intraepithelial cells, show much more junctional diversity (8). In secondary lymphoid organs, T cells using V2, V5, V1.1, and V1.2 are predominant, associated with numerous V chains (2). Again, these chains show substantial junctional diversity. The striking differences between the repertoires of invariant T cells and T cells in the secondary lymphoid organs suggest different functions for these cells. Invariant T cells such as DETCs are thought to recognize predictable self-ligands, which are up-regulated by cell stress such as that associated with wounding or tumorigenesis (9–11). Some of the diverse TCR⁺ T cells in secondary lymphoid organs also recognize self-ligands (12), but others may play a more conventional immunological role by recognizing variable antigens associated with pathogens, consistent with the much greater receptor diversity observed in these populations.

A key question is how such different repertoires of TCR⁺ T cells can be produced during development of the immune system. A striking finding was that invariant V3⁺ and V4⁺ TCR⁺ T cells are only produced in the early fetal thymus (between embryonic days 13 and 17 [E13–17]), whereas the thymus switches to producing only the variable V2⁺, V5⁺, V1.1⁺, and V1.2⁺ populations after E16 (13–15) (for review see reference 16). This specialization is determined by the stage of development of both precursor T cells and thymic stroma (5, 17). The switch has been shown to consist of both programmed events that determine the types of rearrangements that occur at different stages of ontogeny, and a cellular selection process that operates at least in the fetal period to select cells of defined specificity for export to the appropriate peripheral location, such as the skin (for review see reference 18).

Some of the relevant mechanisms are exemplified by the C1 cluster, where the V3 and V4 genes expressed by invariant TCR⁺ T cells are located in the downstream position, closer to J1, whereas the V5 and V2 genes expressed by many variable TCR⁺ T cells are located upstream (Fig. 1 A). The V gene rearrangement pattern changes in ontogeny so that the V3 and V4 genes rearrange preferentially in the early fetal period, whereas the upstream V2 and V5 genes rearrange preferentially in the adult stage (13). Studies with mutated TCR transgenes or TCR knockout mice show that the stage-specific rearrangement pattern is independent of cellular selection processes (16, 19, 20). Rearrangements that occur in the fetal period usually lack N regions (as a result of the absence of terminal deoxynucleotidyl transferase [TdT]) and exhibit little or no junctional diversity (21, 22), whereas the adult-stage rearrangements contain abundant N regions and junctional diversity. On top of these programmed events in development, there is considerable evidence for a selection process that acts in the fetal thymus to select V3⁺ cells that express appropriate V1 chains, and to equip these cells with homing receptors necessary for export from the thymus and emigration to the epidermis (23, 24).

Previous studies of V(D)J rearrangement suggest several possible mechanisms for differential V gene rearrangements. Early studies of V_H genes (25, 26), later extended to V and V gene families (13, 27), showed that rearrangements of downstream V genes are often favored in the fetal period, raising the possibility that the downstream location is inherently favorable for rearrangement at this stage. Later studies showed a correlation between germline transcription of rearranging gene segments and subsequent rearrangements (28, 29) (for review see reference 30). Studies with isolated nuclei showed that the local "accessibility" of rearranging gene segments to exogenously added RAG recombinase is also correlated with subsequent rearrangement (31). The relative roles of transcription versus simple accessibility have proven challenging to dissect, but a recent report provided direct evidence that germline transcription is causally related to rearrangement in the TCR locus (32). Transcription, accessibility, and gene rearrangement have also been linked in some cases to local histone acetylation and/or lack of methylation of gene segments (33, 34). Finally, the specific sequence of a recombination signal sequence (RSS) can restrict its capacity to recombine with other RSS, despite compatibility with the 12/23 joining rule (35). Any or all of these mechanisms may work together or separately to impose the observed pattern of rearrangements.

In previous experimental analyses of the TCR locus, we have focused on the upstream V2 gene as a representative adult-stage gene, and the downstream V3 gene as a representative fetal-stage gene. In adult stage thymocytes, the upstream V2 gene rearranges 25 times more frequently than the downstream V3 gene (20, 36). We implicated the V promoter sequences in this regulation by showing that the preference for V2 over V3 rearrangements in adult thymocytes, as well as the germline transcription pattern, was reversed when the promoter regions of the V3 and V2 genes were swapped in a transgenic backbone consisting of unrearranged V2-4, J1, and C1 gene segments (20, 36). Other studies showed that poor rearrangement of V3 in adult thymocytes correlates with low germline transcription and low levels of histone acetylation (29, 34) of the V3 gene as compared with the V2 gene. Furthermore, inhibitors of histone deacetylase were shown to enhance V3 rearrangements in thymic organ cultures reconstituted with adult bone marrow stem cells (34). These data indicated that rearrangement of V4/V3 genes is strongly repressed in the adult stage by a mechanism that depends at least in part on the V gene promoter segments.

Two lines of evidence suggest that the pattern of rearrangement is regulated by a distinct mechanism in the early fetal thymus. First, although the downstream V3 gene rearranges approximately four times more frequently than the upstream V2 gene in fetal thymocytes (20, 36), both genes were germline transcribed at a relatively high level and showed similarly high levels of histone acetylation (34, 36). Second, swapping the V3 and V2 promoter regions in a transgenic construct did not reverse the fetal pattern of rearrangement, as it did the adult pattern (20). In light of these and additional data with transgenic rearrangement constructs, we proposed that V2 and V3 genes are similarly accessible and transcriptionally active in fetal thymocytes, and that the preference for rearrangement of V3 arises because of an inherent preference for rearrangement of the V gene that lies closest to J1 (18, 36). In addition, we suggested that the preference is established at least in part because of a competition in which the rearrangement of J-proximal V genes inhibits the rearrangement of more distal V genes. In the adult stage, repression of the V3 and V4 genes is proposed to prevent rearrangement of these segments, thus removing the competitive barrier to V2 rearrangement (Fig. 1 B). In this study, we have used a gene targeting approach to directly test two aspects of this proposal in the context of the endogenous TCR locus. The results provide definitive support for the contentions that the downstream V genes enjoy an inherent preference for rearrangement, and that rearrangement of the downstream V genes competitively inhibits rearrangement of the upstream V2 gene segment. Furthermore, both of these effects occur independently of changes in germline transcription, suggesting that the position of the V gene by itself determines the rearrangement pattern in the fetal thymus.

	RESULTS

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Gene-targeted mice to test the role of gene location in programmed V gene rearrangement
To address the role of V gene location in V gene rearrangement in the endogenous TCR C1 locus, we used gene targeting to create a locus with two V2 genes, one in the normal upstream position and the second at the downstream position normally occupied by the V3 gene (Fig. 1 A). This arrangement created the opportunity to examine the rearrangement of essentially identical V genes located in distinct locations in the genome. To create this locus, an endogenous gene segment containing the downstream V4-V3 gene pair was replaced by homologous recombination with an in vitro–assembled V4-V2 pair flanked by loxP sites and a neo cassette, in which the 2.3-kb fragment containing V2 included the V2 promoter region, RSS, and flanking sequences (Fig. 1 A; and Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20071275/DC1). The introduced V2 copy contained a silent single bp substitution that destroyed a KpnI site in the middle of the V2 gene, providing the basis for assays to distinguish this sequence from the wild-type upstream copy when analyzing genomic DNA or mRNA. The loxP sites flanking V4-V2 provided a separate option to delete these V genes in the germline, as described below. After deleting the neo cassette by transfection of embryonic stem (ES) cells with a Cre expression plasmid (Fig. 1 and Fig. S1), we achieved germline transmission of the knock-in (KI) allele, named 242KI. Homozygous 242KI/242KI mice and +/+ littermates were generated by intercrossing (Fig. S1). Because the ES cells were from the 129/SvJ strain, the founder mice were crossed to 129/J mice to minimize genetic diversity and segregation.

Preferential rearrangement of the downstream (J-proximal) V2 gene in the fetal thymus of 242KI/242KI mice
The extent of V gene rearrangement in fetal (E14 or E15) or adult thymocytes was determined by semiquantitative PCR or by Southern blotting. The relative levels of rearrangement of the two V2 genes were determined by digesting the PCR products with KpnI (Fig. 2 A, left), or by performing Southern blots with DNA digested with restriction enzymes that allowed the two rearrangements to be distinguished by size (Fig. 2 B). In E14 fetal thymocytes, the downstream V2 gene segment rearranged four to five times more frequently than the upstream V2 gene. In E15 fetal thymocytes, the difference decreased to twofold (Fig. 2, A and B). Accordingly, the total level of V2 rearrangements (the sum of rearrangements of the upstream and downstream V2 genes) was elevated approximately fourfold in the 242KI E14 fetal thymocytes compared with wild-type fetal thymocytes (Fig. 2 C). The substituted V2 gene in the downstream position did not detectably alter the extent of V4 rearrangements (unpublished data).

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Figure 2. V2 gene rearrangements in 242KI/242KI and wild-type (+/+) thymocytes. (A) PCR analysis of rearrangements of upstream versus downstream V2 genes in E14 fetal and adult thymocytes. Radiolabeled PCR products were amplified from thymocyte DNA with primers L2 and J1, digested with KpnI, and subjected to electrophoresis and PhosphorImager analysis (as described in Materials and methods). Parallel PCR analysis of wild-type genomic DNA was included to demonstrate complete KpnI digestion of PCR products of the wild-type (upstream) V2 gene. (left) Thymocytes from Tcr+/+ mice. (right) Thymocytes from TCR–/– mice, to examine rearrangement under conditions in which TCR receptors cannot be expressed. The 242KI mice in this experiment only had been backcrossed to C57BL/6J. (B) Southern analysis of V2 gene rearrangements in BamHI/NcoI-digested adult thymocyte and liver DNA hybridized with a V2 probe. Bands corresponding to rearranged (rr) and germline upstream (endogenous) and downstream (knocked-in) V2 genes are indicated. The ratio of downstream/upstream V2 rearrangements was 2.1-fold in E15 fetal thymocyte DNA, compared with 0.4-fold in adult thymocytes. Vertical black lines in A and B indicate lanes deleted in construction of the figure. (C) Semiquantitative PCR analysis of total V2 gene rearrangements (the sum of upstream and downstream [if present] V2 rearrangements) or control tubulin sequences in E14 fetal or adult thymocyte samples. V2 was rearranged fourfold more often in 242KI than in wild-type E14 fetal thymocytes, but there was no difference in adult double-negative thymocytes.

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The four- to fivefold preference for rearrangement of the downstream V2 gene in 242KI/242KI mice is comparable to the approximately fourfold preference for rearrangement of the downstream V3 gene observed in wild-type fetal thymocytes (20, 36). Therefore, the location of the V2 gene segment in the V gene array determines its preference for rearrangement in the fetal period independently of the RSS or the upstream promoter regions, which were identical in the two V2 genes under comparison.

Deletion of V4 and V3 results in increased rearrangement of V2 at the fetal stage
Preferential rearrangement of the J-proximal V gene at the fetal stage might reflect a competition between V genes for rearrangement to J1, in which the downstream V gene holds an advantage. If this were the case, deletion of the downstream V genes should result in an increased frequency of rearrangements of the upstream V gene. Deletion of the downstream V genes was possible because the V4-V2 gene pair in the targeted locus was flanked by loxP sites. Among the Cre-transduced ES cell colonies, we identified some in which this entire segment, as well as the neo cassette, were deleted by Cre-mediated recombination (Fig. 1 and Fig. S1). The deletion reduced the distance between V2 and J1 by only a modest 20% compared with the wild-type locus. The knockout allele was named 43 to signify the fact that this targeted allele corresponds to a deletion of the V4 and V3 genes relative to the wild-type locus. After generating chimeras with the recombinant ES cells and achieving germline transmission of the 43 allele by crossing to 129/J mice, homozygous 43/43 mice were generated by intercrossing.

The amount of V2 gene rearrangement to J1 in 43/43 mice was compared with that in wild-type (+/+) littermates on the 129 background. As shown by semiquantitative PCR, the deletion of V3 and V4 resulted in an approximately fivefold increase in V2 rearrangement in E14 fetal thymocytes (based on three experiments; Fig. 3 A and not depicted). Thus, deletion of V3 and V4 resulted in increased levels of V2 rearrangement in E14 fetal thymocytes to about the level of V3 rearrangement observed in wild-type mice. Comparable PCR analysis (not depicted) and genomic Southern blotting with a V2 probe (Fig. 3 B and not depicted) revealed that V2 rearrangements were still elevated in 43/43 mice on E15, though to a lesser extent. A separate examination of rearrangements of V5, which is also upstream of V3/V4, showed an approximately threefold increase in 43/43 mice compared with wild-type mice in E14 fetal thymocytes (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20071275/DC1). These data indicated that rearrangements of the proximal V3 and V4 genes in the wild-type locus normally competitively inhibit rearrangements of the more distal V2 and V5 genes.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 3. V2 gene rearrangements in 43/43 mice. (A) Two experiments examining V2 rearrangements in E14 fetal thymocytes of wild-type and 43/43 mice by semiquantitative PCR. The PCR for tubulin was used as a loading control. (B) Southern blot analysis of V2 gene rearrangements in BamHI/NcoI-digested adult thymocyte or liver genomic DNA hybridized with a V2 probe. Bands corresponding to germline and rearranged (rr) V2 genes are indicated. The percentage of rearranged/(rearranged + unrearranged) V2 alleles in each lane is presented at the bottom.

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View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 4. V2 gene germline transcription and restriction enzyme accessibility in 242KI mice. (A) Comparison of germline transcripts of the upstream versus downstream V2 gene in E14 fetal thymocytes and adult double-negative (DN) thymocytes from 242KI/242KI mice by semiquantitative RT-PCR (see Materials and methods). The PCR products were digested with KpnI before electrophoresis and PhosphorImager analysis to distinguish their origin from the downstream (uncleaved) versus upstream (cleaved; note that the smaller fragment has run off the gel) V2 genes (see Materials and methods). Semiquantitative RT-PCR for tubulin was used as a loading control. The ratios of downstream/upstream V2 rearrangements are depicted below the panels (means of two assays). Vertical black lines indicate lanes deleted in construction of the figure. –RT, sample without RT. (B) Accessibility to ScaI cleavage of ScaI restriction enzyme sites located upstream of both V2 gene copies in the nuclei of 242KI E15 fetal thymocytes. The bands corresponding to upstream and downstream V2 genes that were accessible (cleaved) versus inaccessible (uncleaved) to ScaI are indicated on the right, along with the percentage of cleavage.

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We also used a restriction enzyme accessibility assay to determine whether the two copies of the V2 gene were differentially accessible in the nuclei of E15 fetal thymocytes from 242KI/242KI mice. A ScaI restriction enzyme site 380 bp upstream of the V2 ATG codon was nearly equally accessible to restriction enzyme in the two copies of V2 in 242KI/242KI mice in the nuclei of E15 fetal thymocytes (Fig. 4 B). Therefore, the different levels of rearrangement did not correlate with the accessibility of this site to restriction enzyme.

To assess whether the increased rearrangement of V2 in fetal thymocytes from 43/43 mice could be attributed to greater germline transcription, we quantified V2 germline transcripts in 43/43 versus +/+ E14 and E15 fetal thymocytes. In two experiments, fetal thymocytes from the two strains contained essentially identical levels of V2 germline transcripts (Fig. 5 and not depicted). Thus, the substantial increase in V2 rearrangements that accompanied deletion of V4 and V3 is not accompanied by a corresponding increase in V2 germline transcription.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Germline transcription of V2 and J1 genes in 43/43 versus wild-type fetal thymocytes. E14 and E15 fetal, and adult CD4–CD8– thymocyte RNA samples were reverse transcribed with random hexamer primers and subjected to semiquantitative PCR analysis with V2 or J1 primer sets to detect germline transcripts (see Materials and methods). Two replicate samples were run in parallel in some cases, as indicated. The ratios of V2 germline transcripts in wild-type mice versus 43/43 mice, calculated from PhosphorImager data and normalized with the PCR for β-tubulin transcripts, were 0.9 for E14 fetal thymocytes, 1 for E15 fetal thymocytes, and 1 for adult thymocytes (mean of at least two samples for each). The ratios of J1 germline transcripts in wild-type versus 43/43 mice were 0.7 for E14 fetal thymocytes, 1 for E15 fetal thymocytes, and 1.1 for adult thymocytes (mean of at least two samples for each). Samples lacking RT (–RT), run in parallel, yielded no PCR products, as shown for adult thymocytes (bottom). Vertical black lines indicate lanes deleted in construction of the figure.

Influence of V gene location on the rearrangement and accessibility of V genes at the adult stage

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We also determined whether the decreased proportion of downstream V2 rearrangements at the adult stage was paralleled by a decrease in the proportion of germline transcripts emanating from the downstream gene. The analysis showed a reduction of approximately threefold in the proportion of germline transcripts emanating from the downstream gene (Fig. 4 A). These data must be adjusted based on the relative number of copies of each unrearranged V2 gene in the population being examined. A separate PhosphorImager analysis (see Materials and methods) of three Southern blots, examining band intensity in thymocytes as compared with liver DNA, showed that the ratio (downstream/upstream) of unrearranged V2 genes in adult thymocytes was 0.74 (unpublished data). Hence, the abundance of V2 germline transcripts emanating from the downstream V2 gene was reduced by 2.2-fold after normalization for the number of gene templates in the population. The decreased proportion of rearrangements and germline transcripts of the downstream V2 gene segment in adult thymocytes suggests that sequences in the vicinity of the downstream gene repress both germline transcription and rearrangement at the adult stage. Because the downstream V2 gene segment is identical to the upstream segment, the relevant repressive sequences are presumably not in the downstream V2 gene segment itself, but more likely in neighboring sequences. Hence, these data are consistent with the existence of sequences in the downstream region in addition to those previously attributed to the V3 promoter (which is absent from the 242KI chromosome) (20), which locally repress rearrangements in the adult thymus.

In adult thymocytes, rearrangements of the upstream V2 gene were similarly abundant in wild-type mice as in either 242KI/242KI (Fig. 2 B) or 43/43 (Fig. 3 B) mice. Furthermore, in adult thymocytes, V2 germline transcripts were similarly abundant in wild-type, 242KI/242KI (Fig. 4 A, legend), and 43/43 (Fig. 5) thymocytes. Hence, the alterations at the downstream position in these two chromosomes did not alter the efficiency of rearrangements or germline transcription at the upstream position in adult thymocytes.

	DISCUSSION

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Previous studies (20, 36) demonstrated that the downstream V3 gene is preferentially rearranged in the early fetal thymus compared with V2 by a factor of approximately fourfold. Other studies show that the fraction of productive rearrangements is inherently lower for V2 than for rearrangements of other V genes, because V2 uniquely contains an in-frame stop codon near its 3' end, which must be removed (presumably by endonuclease action) during the recombination process (16). The relative paucity of V2 rearrangements at the fetal stage, coupled with the rarity of productive V2 rearrangements, are critical in ensuring that nearly all TCR⁺ fetal thymocytes express V3 (or V4) and not V2. The absence of TdT at the fetal stage also plays a role in the process of V3 gene rearrangement. Without TdT and the N nucleotides it adds, the frequency of rearrangements that occur at the site of a dinucleotide homology shared by the V3 and J1 gene segments is increased, thus ensuring that most productive rearrangements carry precisely the same (canonical) V-J junctional sequence (22). These mechanisms, coupled with a selection process for V3⁺ T cells that operates in the fetal thymus (24), conspire to convert the early fetal thymus into an organ specialized in part for the production of V3⁺ DETCs.

Our data using gene-targeted chromosomes address a key part of this coordinated process by probing the mechanisms that lead to preferential V3 gene rearrangement in the fetal thymus. The results have implications for other Ig/TCR genes as well, because preferential rearrangement of J-proximal V genes is also observed in early stage development of B cells and T cells in the case of the IgH and TCR gene families. The results of this study provide compelling in vivo evidence that the location of a V gene is a critical determinant of its rearrangement pattern in the fetal thymus and provide direct evidence that the downstream gene exerts competitive inhibition of upstream V gene rearrangement at the fetal stage.

Previous studies provided evidence that at the fetal stage, the unrearranged V2 and V3 gene segments are acetylated on histone residues to a similar extent, and are germline-transcribed similarly as well (34, 36). These similarities suggested that neither accessibility nor transcriptional activity of the genes was a likely explanation for the preferential rearrangement of the downstream V genes, raising the possibility that gene location by itself plays a role in rearrangement at the fetal stage. This interpretation was further suggested by our previous study performed with transgenes consisting of unrearranged V/J/C genes, in which the positions of the V3 and V2 gene segments were swapped within the transgene (36). The present study, however, is a definitive analysis, as it was performed by targeting the endogenous locus, and is therefore free of certain artifacts that can confound interpretation of transgenic mice, such as the existence of multiple tandem copies of the transgene that create the opportunity for complex rearrangements to take place and that may override natural regulatory processes, as well as the potential absence of flanking regulatory sequences that may influence the rearrangement process. Equally important, the current study is superior as it examines two copies of the same complete V gene segment (V2) located at the upstream and downstream positions. Hence, differences in rearrangement cannot be attributed to differences in the RSS or promoters of the genes, or other features associated with the coding and immediately flanking DNA, all of which were included in the inserted downstream V2 copy.

The results showed that the downstream V gene enjoyed an approximately fivefold advantage in rearrangement frequency in the early fetal thymus. The upstream and downstream V genes were germline transcribed to a similar extent, suggesting that the advantage did not result from greater accessibility or transcription-related enhancement of rearrangement. We propose, instead, that the advantage of the downstream V gene arises from a greater likelihood of encountering the J1 RSS, either because of its closer proximity or because of a processive aspect to the rearrangement process that favors the first encountered V gene. We cannot rule out the possibility that the advantage arises from closer proximity of the downstream gene to a downstream regulatory element. However, we previously showed that rearrangement is not significantly altered in the fetal thymus of mice with targeted deletions of either or both of the two enhancer-like elements in the locus, including the downstream enhancer E_3'C1 (37).

Based on these findings, we hypothesized that the fetal pattern of rearrangement is determined by a competition between the downstream and upstream V gene segments, with an advantage accruing to the downstream V gene owing to its closer proximity to J1 (Fig. 1 B). The design of our gene-targeted allele enabled us to test for the first time a key prediction of this hypothesis by determining whether deletion of the intervening V4 and V3 genes altered the putative competition so as to promote rearrangement of the upstream V2 gene. The results demonstrated that deletion of V4 and V3 resulted in enhanced rearrangements of the upstream V2 gene segment in the fetal thymus to an extent commensurate with the amount of rearrangement that normally occurs at the downstream V gene. These findings provide direct support for the notion that V genes are normally in competition in the fetal stage, and that J-proximal rearrangements occur at the expense of J-distal rearrangements. The small reduction in the length of the segment of DNA connecting V2 and J1 resulting from the deletion, 20%, appears unlikely to account for an approximately fivefold increase in rearrangement in E14 fetal thymocytes.

As discussed in the Introduction, the pattern of rearrangement in the adult thymus is imposed by a distinct mechanism that is regulated by V promoter–specific sequences and leads to repression of V3 and V4 rearrangements that is dependent on local histone deacetylation. Considering all these observations, we proposed that the V3 promoter contains sequences that locally repress V3 and V4 rearrangements in the adult, abolishing competition from downstream genes and enhancing upstream V gene rearrangements (18). The notion that V3-associated sequences repress rearrangement in the adult thymus predicts that replacing V3 and its promoter with corresponding V2 sequences, as in the 242KI mice, should result in less effective silencing of downstream rearrangements in adult thymocytes. The data are consistent with this interpretation, because the bias in favor of rearrangement of the upstream V2 gene in adult 242KI/242KI mice (2-fold) was small in comparison to the bias in favor of V2 over V3 rearrangements in adult wild-type mice (25–50-fold). On the other hand, although the overall level of rearrangement increased substantially between the fetal and adult stages, presumably because of enhanced overall activity of the locus, the relative abundance of downstream V2 rearrangements in adult thymocytes of 242KI/242KI mice declined by 10-fold compared with the situation in fetal thymocytes. A plausible explanation for these data is that the 242KI allele retains residual repressive sequences active at the adult stage. One possibility is that the V4 gene, which remains in the 242KI allele, contains these putative residual repressive sequences.

The results reported in this paper support a significant role of gene position and V gene competition in controlling the abundance of specific V gene rearrangements. These features must therefore be considered in conjunction with transcriptional activity, accessibility, differences in RSS sequences, and other factors as important regulators of V(D)J recombination. All of these factors may be exploited in the evolution of mechanisms to create various specific repertoires in the adaptive immune system. This is illustrated by the dynamic behavior of the TCR locus during development, in which the positional influence, gene competition and other factors work in tandem to enable the early fetal thymus to specialize in the production of invariant TCR⁺ T cells destined for epithelial locations, whereas transcriptional repression and/or loss of accessibility enable the adult thymus to switch its efforts toward the production of V2⁺ and other TCR⁺ subsets found abundantly in secondary lymphoid organs in adult mice.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of 242KI and 43 knockout mice.
A single gene targeting event was used to generate 242KI and 43 knockout mice. The targeting vector (Fig. 1 A and Fig. S1) was based on the pKSTKNeoLoxP plasmid (a gift from R. Rickert, Burnham Institute for Medical Research, La Jolla, CA). It included a thymidine kinase cassette at the 5' end, a 5' homology region corresponding to a 3.2-kb SnaBI-BglII fragment upstream of the V4 gene, and a 3' homology region corresponding to a 3.2-kb EcoRI-BamHI fragment downstream of the V3 gene. Between the arms, we inserted a PGK-neo cassette and a V4-V2 gene pair comprising a 1.7-kb BglII-HindIII V4 fragment ligated to a 2.5-kb SpeI-EcoRI V2 fragment. The latter fragment was the same fragment used in an earlier transgenic study (20, 36), and it included the V2 promoter, leader exon, intron, coding segment, RSS, and 3' flanking DNA. In the present study, the inserted V2 fragment contained a silent 1-bp mutation that destroyed the unique KpnI site located in the coding region of this gene (ggtaccggtatc) to enable discrimination from the upstream wild-type gene. The PGK-neo cassette was flanked by loxP sites, and an additional loxP site was located downstream of V2 at the 3' end of the inserted segments (Fig. 1 A). The locations of the three loxP sites enabled us to delete the PGK-neo cassette or the entire inserted sequence, depending on whether the 5' loxP site recombined with the central loxP site or the 3' loxP site.

The targeting construct was transfected into J1 ES cells of the 129/SvJae strain (38), followed by selection with G418 and ganciclovir. Targeted ES cell clones were identified by Southern blotting with a 5' flanking probe (Fig. S1 A). Correct targeting at the 3' end was confirmed by PCR (Fig. S1 B). The targeted ES cell clones were transiently transfected with a Cre-expressing plasmid (39) and screened by Southern blotting for clones that had deleted only the PGK-neo cassette (242KI), or had deleted both the PGK-neo cassette and the V4-V2 fragment (43; Fig. S1 D). The recombinant ES cell clones were separately injected into C57BL/6 blastocysts, and chimeric mice were crossed with 129/J and C57BL/6J mice. Heterozygous pups from the 129 cross were intercrossed to generate homozygous 242KI/242KI or 43/43 mice on the 129 background. Heterozygous pups from the C57BL/6J cross were serially backcrossed to C57BL/6J mice six times before intercrossing. Blotting genomic DNA with V3, V2, and other probes confirmed germline transmission of the targeted alleles (Fig. S1 E and not depicted). Animal studies were approved and overseen by the University of California, Berkeley Animal Care and Use Committee.

Cell, DNA, and RNA preparations.
Total and CD4^–CD8^– (double-negative) thymocytes were prepared as previously described (37). DNA and total cellular RNA were prepared as previously described (20).

Primers.
Primers L2, J1, PSV2, VG2I, V2-3'a, 5'J1 5'-2, C1 3'-3, 5' tubulin, and 3' tubulin have been previously described (18, 20, 29, 34, 36). Primer PSV2' is as follows: tcgaaagctttaggagtgtaacaatacac.

Semiquantitative PCR and RT-PCR analysis.
The semiquantitative PCR assays were performed essentially as previously described (20). In brief, thymic genomic DNA was subjected to PCR in the presence of -[³²P]deoxycytidine triphosphate (dCTP) with the primer set L2/J1 to amplify V2 gene rearrangements. To distinguish whether the radiolabeled PCR products originate from rearrangements of the endogenous upstream V2 gene versus the knocked-in downstream V2 gene in 242KI mice, they were digested with Kpn1 before electrophoresis on a polyacrylamide gel and analysis (PhosphorImager; Molecular Dynamics).

Semiquantitative RT-PCR performed as previously described (18, 36), with minor modifications, was used to quantify V2 or J1 germline transcripts. RNA samples from E14 fetal thymocytes or adult CD4^–CD8^– thymocytes were treated with DNase to eliminate any genomic DNA contamination and reverse transcribed with Superscript II RNase H^– RT using random hexamer primers. The RT products were serially diluted (by the factors indicated in the figures) and subjected to PCR in the presence of -[³²P]dCTP with primer sets PSV2/V2-3'a (for analysis of V2 transcripts in 242KI mice), PSV2'/V2-3'a (for analysis of V2 transcripts in 43 mice), or 5'J1 5'-2/C1 3'-3 (for J1). In the case of samples from 242KI mice, the RT-PCR products (307 bp) were digested with Kpn1 to distinguish the origin of the products. RT-PCR products corresponding to the mutated 3' V2 copy cannot be cleaved by KpnI, whereas products corresponding to the endogenous 5' V2 gene are cleaved into smaller fragments (274 + 33 bp). Parallel semiquantitative RT-PCRs of β-tubulin transcripts were used to normalize the samples. In all cases, RNA samples without RT were subjected to PCR in parallel to assure that there was no genomic DNA contamination.

Southern blot analysis of V2 gene rearrangements.
Thymic genomic DNA samples were digested with NcoI/BamHI and analyzed by Southern blotting using a V2-specific probe, as previously described (20).

Restriction enzyme accessibility assay.
The assay was performed to assess restriction enzyme accessibility of the two copies of V2 genes in 242KI mice. In brief, nuclei were prepared from E15 fetal thymocytes and digested with ScaI for 1 h. After the ScaI digestion, DNA was prepared from the nuclei and digested with NcoI. The DNA was analyzed by Southern blotting using a V2 probe.

Online supplemental material.
Fig. S1 depicts and confirms the gene targeting procedure used to generate 242KI and 43 mice. Fig. S2 shows changes in the rearrangement of V5 in 43 mice. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20071275/DC1.