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High-Level Rearrangement and Transcription of Yeast Artificial Chromosome-Based Mouse Ig Transgenes Containing Distal Regions of the Contig¹

Shuyu Li^*, Robert E. Hammer, Julia B. George-Raizen^*, Katherine C. Meyers^* and William T. Garrard^2,*

^* Department of Molecular Biology and Oncology and Biochemistry, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75235

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mouse Ig L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon V-J joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream V2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream V21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Ig miniloci that contained differing lengths of sequences 5' of the most distal V2 gene family member. In transgenic mice, Ig miniloci exhibited position-independent and copy number-dependent germline transcription. Ig miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal V gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5' region exhibited significantly greater recombination of the upstream V2 genes but not the downstream V21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Ig gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mouse Ig gene locus has provided a paradigm to study many interesting biologically relevant problems, including: DNA sequence organization and evolution (1), tissue-specific transcriptional regulation (2, 3), site-specific recombination (4, 5, 6), somatic hypermutation (7, 8, 9), various aspects of DNA methylation (10, 11, 12), and the relationship between chromatin structure and function (13, 14, 15, 16, 17).

The mouse locus is the largest Ig gene locus thus far identified, spanning more than 3.5 Mb (1, 18, 19). The locus contains 93 potentially functional V regions that have been grouped into 18 families based on sequence homologies, four functional and one nonfunctional J regions, and a single C exon (20, 21, 22, 23, 24, 25). The most 5' V region family was originally thought to be V2, 3.5 Mb away from the J-C region (18, 19). However, recently a single V24 family member has been found further upstream (23, 24, 25). The most 3' V region family is V21, and the closest V21 gene member is V21G, 18 Kb away from J1 gene segment (26).³

Several cis-acting regulatory elements have already been identified in the mouse Ig locus. Except for V region promoter elements, all of these elements reside near or within the J-C region at the 3' end of the locus. These include: two germline promoters (27), KI-KII sequences (28), a nuclear matrix association region (MAR)⁴ (29), an intronic enhancer (30), and a 3' enhancer (31). Targeted deletions of these elements in cells or mice have been performed, allowing their functional significance to be addressed in the native locus. Targeted deletion of the KI-KII sequences, a germline promoter, or both reveals a phenotype of suppressed recombination (28, 32, 33). Targeted deletion of the MAR in cultured pre-B cells results in hyperrecombination (11), whereas deletion of the same element from the mouse germline only mildly advances the timing of V-J joining during development (34). Deletion of either the intronic or 3' enhancers severely affects Ig gene rearrangement but does not abolish it (35, 36), suggesting either that each enhancer can partially compensate for the loss of the other or that other elements also contribute to Ig gene expression.

Despite identification of all of the above regulatory elements, the results of transgenic mice studies strongly suggest that additional crucial regulatory elements within the Ig locus remain to be discovered. Human Ig germline minilocus transgenes containing all the corresponding known regulatory elements described above have been ectopically introduced into the mouse germline. Only poor and erratic expression was noted relative to that obtained by the endogenous mouse Ig locus (37, 38, 39, 40, 41). However, because more recent studies have been successful in achieving high-level expression of human Abs after introducing either entire chromosomes or 300-1300 kb yeast artificial chromosomes (YACs) bearing the human Ig locus into mice (42, 43), it is likely that other important regulatory elements present in the native human locus but not present on the earlier shorter transgene constructs remain to be discovered. The missing sequences may reside in the distal 5' and 3' regions of the locus, because the above poorly expressed transgenes did not possess both of these regions on one construct. We propose a similar case for the mouse locus. While sequences centrally located in the locus are frequently deleted upon V-J joining, the upstream and downstream regions are always maintained in a recombined native locus.

YAC technology has provided an ideal tool to manipulate and engineer large DNA fragments. YAC clones have been introduced into transgenic mice and mammalian cells to study gene expression (44, 45). In this report, we have used YAC-based technology to fuse 5' and 3' distal regions of the mouse Ig locus to create 225–290 kb Ig miniloci with differing lengths of sequence 5' of the V2 gene cluster. We have generated transgenic mice carrying these miniloci and functionally analyzed their behavior in germline and rearranged gene transcription as well as V-J joining. This approach has for the first time achieved high levels of both rearrangement and transcription of rearranged mouse Ig transgenes, suggesting that most regulatory elements in the locus reside within our Ig miniloci. These miniloci should be valuable as reagents for future functional analyses of cis-acting elements.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Retrofitting YAC FAR.E8 vector arms

The selectable markers TRP1 and URA3 on the vector arms of the YAC clone FAR.E8, which resides in the 5' region of the contig (18), were replaced with LYS2 and HIS3, respectively, by one-step gene replacement (46). The TRP1-targeting vector contained a new selectable marker, LYS2, flanked by fragments of the gene TRP1. It was constructed as follows: an EcoRI and ClaI fragment containing LYS2 was excised from pDA6200 (47) and subcloned into pBluescript (Stratagene, La Jolla, CA) as pSK/LYS2. The 5' fragment of TRP1 was amplified by primers (5' to 3') GCT CTG CAG TGG AAA ACG TTC TTC GGG GCG and CTT GAA TTC TAT TGA AAA AGG AAG AGT ATG using pRS304 (Stratagene) as template, and it was subcloned into the PstI and EcoRI sites of pSK/LYS2. The 3' fragment of the TRP1 was PCR amplified by primers (5' to 3') CTG CAT CGA TAT GAG TCG TGG CAA and AAC CCA GTC GAC AAT CGA GTT CCA ATC CAA from pRS304 and subsequently cloned into the ClaI and SalI sites of pSK/LYS2. The PCR amplifications were conducted for 1 min at 94°C, 2 min at 55°C, and 1 min at 72°C for 30 cycles. The resulting construct was digested with PstI and SalI to release targeting sequences. Then, 1 µg of targeting DNA fragments was transformed into FAR.E8-bearing yeast cells by the lithium acetate method (48). After selecting for growth on medium lacking lysine, targeted transformants were confirmed by PCR and Southern analyses.

Similarly, the URA3-targeting vector contained a new selectable marker, HIS3, flanked by fragments of the gene URA3. The 5' and 3' fragments of URA3 were PCR amplified from pRS306 (Stratagene). The primers (5' to 3') for the 5' fragment were AGG AGC TCG AGT CGA AAG CTA CAT ATA AGG AAC GTG and GAT TTT TCC ATG GAG GGC ACA GTG CGG CCG CTA; primers for the 3' fragment were GCA GAA TTC TCA TGC AAG GGC TCC CTA GC and GTG GAT GAT GTG GTC TCT ACA GGA CTC GAG ATT. To construct the HIS3::ura3 vector, a BamHI fragment containing the HIS3 gene from pRS303 (Stratagene) was subcloned into pBluescript, followed by subcloning of the 5' and 3' URA3 fragments into SacI/NotI and EcoRI/XhoI sites, respectively. The targeting fragment was released by digestion with XhoI and was transformed into FAR.E8-bearing yeast cells. The transformants were selected for growth on medium lacking histidine and confirmed for targeting by PCR and Southern analyses. Retrofitted FAR.E8 was analyzed by pulsed-field gel electrophoresis (PFGE) and Southern hybridization to confirm that no obvious deletion or aberrant recombination occurred during these processes.

Chromosome fragmentation of YAC FAW.A3

Chimeric material in the YAC clone FAW.A3, which resides within the 3' region of the contig (18), was eliminated by chromosome fragmentation (46). The fragmentation vector was constructed as follows. First, a SalI/ClaI yeast telomere fragment was subcloned into pRS315 (Stratagene) containing the LEU2 selectable marker, CEN and ARS sequences. Then a BamHI fragment from plasmid pRSB (49), which contains the cryptic recombination sequence (RS), was subsequently subcloned into pRS315. The targeting vector was linearized by SmaI and transformed into yeast cells bearing FAW.A3. Transformants were selected by growth on synthetic medium lacking leucine. Those transformants resulted from self-ligation and propagation of the targeting vector were eliminated by replica plating on medium lacking both leucine and tryptophan. Final candidates were analyzed by PFGE.

Generation of the ML by "end fusion"

We purified retrofitted FAR.E8 and FAW.A3d after PFGE under conditions of mild shearing for cotransformation. This resulted in DNA fragments in the range of 200–300 kb that were highly recombinagenic. The PFGE run conditions were 1% SeaPlaque agarose (FMC BioProducts, Chicago, IL), 0.25x TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.3), auto algorithm separating DNA with a size range of 400–800 kb, using a Bio-Rad CHEF mapper apparatus (Bio-Rad, Richmond, CA). The gel slices corresponding to the YACs were excised and equilibrated in 1x TAE (40 mM Tris-acetate, 1 mM EDTA, pH 7.8), 100 mM NaCl, 30 mM spermine, and 70 µM spermidine for 2 h at room temperature. Gel slices were melted at 68°C for 10 min and treated with Gelase (Epicentre Technologies, Madison, WI) at 1 U/100 mg of gel at 42°C for 2 h. The DNA of both YACs (1 µg each) was cotransformed into the host strain YPH857 by spheroplast transformation using 10⁹ cells (50). Yeast cells bearing potential recombinant YACs were selected for by the presence of the two external markers, HIS3 and LEU2, for growth in the corresponding dropout medium, and for the absence of the internal markers, URA3 and LYS2, by negative selection with 5-fluoroorotic acid and -aminoadipic acid. We screened 11 clones of recombinant YACs by PCR for the presence of V2 and V21, the most 5' and 3' V gene families, and identified 7 YACs positive for both V families. Further analysis by PFGE as above revealed a 240-kb candidate Ig minilocus (ML) that had an ideal size for future transgenic and cell culture experiments.

Indirect end-labeling assay of ML

Total DNA from yeast cells carrying the ML, or one of the parent YACs, FAR.E8 or FAW.A3, was partially digested with SfiI and separated by PFGE. DNA was nicked by exposure to UV light (180 mJ) and transferred to Nytran-Plus membrane, pore size 0.22 µM (Schleicher & Schuell, Keene, NH). The DNA was hybridized to either of the vector arm probes, L or R, labeled with ³²P using a Random Primer Labeling kit (Amersham, Arlington Heights, IL). Hybridization was conducted as previously described (51). The hybridization was at 65°C for 18 h. Filters were then washed with 0.2x SSC, 2% SDS for 30 min and 0.1x SSC, 2% SDS for 30 min at 65°C. Membranes were exposed to PhosphorImaging screens, and images were analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Generation of single-base changes in J and C regions in the ML

The procedure used to create single-base changes has been described previously (52). The Kluvermyces lactis URA3 (kURA3) gene was first amplified from genomic DNA using primers with 50-bp tails that result in a direct repeat spanning the desired mutation. The primers (5' to 3') for the J-targeting construct were AAC TAG GGG AAG AGG GAT AAT TGT CTA CCA TGG GAG GGT TTT GTG GAG GTA GCT CTT CAA TTC ATC TTT TTT TTT TTT GTT CTT T and ACC TCC ACA AAA CCC TCC CAT GGT AGA CAA TTA TCC CTC TTC CCC TAG TTG GGT AAT AAC TGA TAT AAT TAA ATT GAA GCT. Each primer was used for single-strand extension using K. lactis genomic DNA as template for 15 cycles at 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min. Both amplification reactions were mixed, and 20 cycles of amplification were conducted under the same conditions. To increase the efficiency of targeting, the resulting PCR product was reamplified by adding 70 bp of flanking sequence to each direct repeat. The primers were (5' to 3') GAA AAC TGT CCC ACA AGA GGT TGG AAT GAT TTT CAG GCT AAA TTT TAG GCT TTC TAA ACC AAA GTA ACT AAA CTA GGG GAA GAG GGA TAA TTG TC and ATG TAC TTA GGT TTT ATT TCC AGT CTG GTC CCA TCA CTG AAT GTG ATT TAC AGT GAT TTA TTT TAA CTT TAC CTC CAC AAA ACC CTC CCA TGG TA. The amplification was performed as described above.

The primers (5' to 3') for the C-targeting construct were ACG ACA AAA TGG CGT CCT GAA CAG TTG GAC AGA TCA GGA CAG CAA AGA CAG CAC CTA CAG CAT GAG CAG GTG ATT CTG GGT AGA AGA TCG GTC and CTG CTC ATG CTG TAG GTG CTG TCT TTG CTG TCC TGA TCT GTC CAA CTG TTC AGG ACG CCA TTT TGT CGT TGT GTG CTT GCT TCT TTT CTT ATC CG. Similarly, the PCR product was reamplified by adding 70 bp flanking sequence to each direct repeat. The extension primers (5' to 3') were AGT CGT GTG CTT CTT GAA CAA CTT CTA CCC CAA AGA CAT CAA TGT CAA GTG GAA GAT TGA TGG CAG TGA ACG ACA AAA TGG CGT CCT GAA CAG and GTC TTG TGA GTG GCC TCA CAG GTA TAG CTG TTA TGT CGT TCA TAC TCG TCC TTG GTC AAC GTG AGG GTG CTG CTC ATG CTG TAG GTG CTG TCT. The conditions for PCR reactions were as described above. Then, 1 µg each of targeting constructs were transformed into ML-bearing yeast cells, and transformants were selected for the kURA3 gene and then screened for targeting by PCR. Next, negative selection with 5-fluoroorotic acid was conducted to isolate cells that had spontaneously looped out the kURA3 gene. PCR amplification and digestion by AvrII/NcoI or by BclI were used to diagnose whether the introduced mutations were retained after recombination.

Generation of a 5'-deleted minilocus (5'DML) by chromosome fragmentation

5'DML was created by eliminating the 15-kb upstream sequence from ML by chromosome fragmentation. The fragmentation vector was constructed as follows. A ClaI/SalI telomere fragment from pBP103 (American Type Culture Collection, Manassas, VA) was cloned into pRS304 (Stratagene). Targeting sequences were amplified by PCR using a V2(70/3)-specific primer pair engineered to have a PmeI site at its 3' end followed by SalI sites at both ends. The primers were (5' to 3') ACG CGT CGA CAG GAA GCC CAC ATA ACT GCC CCT, and ACG CGT CGA CGT TTA AAC TCC AGA GTC CAG TTT AGA CAC CAG A. The PCR conditions were 1 min at 94°C, 2 min at 55°C, and 1 min at 72°C for 30 cycles. The SalI cleaved products were gel purified and cloned into pRS304. The final vector was linearized by PmeI and transformed into ML-bearing yeast cells. Transformants were selected for the presence of the TRP1 marker and screened for lack of the HIS3 marker by replica plating.

Construction of a 5'-extended minilocus (5'EML)

A 684-bp single copy DNA fragment designated P5 was amplified from a region located 3.5 kb upstream of V2(70/3). The primers (5' to 3') were GTC TCT TCT AGC CCA CTG ACC ATA G and CTT GAA TGG CCA AGA AGC ACT TAG AGA. The PCR amplifications were conducted 1 min at 94°C, 2 min at 55°C, and 1 min at 72°C for 30 cycles. The P5 fragment was used as a probe in screening the mouse genomic BAC library from Research Genetics (Huntsville, AL). A 100-kb BAC clone (designated 5'BAC) was identified that hybridized to the P5 probe. 5'BAC DNA was digested with NotI, and a 90-kb fragment spanning the majority of the BAC insert plus 200 bp of the BAC vector sequence was gel purified after PFGE. The NotI fragment was ligated to a linker plasmid containing a telomere seed fragment, a neomycin resistance gene, and the TRP1 selectable marker, followed by digestion of 500 ng DNA with 6 U of exonuclease III for 10 min at 37°C. The resulting fragment was transformed into ML-bearing yeast cells by spheroplast transformation, and transformants were selected for the TRP1 marker. After replica plating, those transformants that had lost the HIS3 marker were analyzed by PFGE.

Southern hybridization analyses of the 5'EML

First, 20 ng of BAC DNA and 3 µg of total yeast DNA isolated from the YPH857 yeast strain, or the same strain bearing either ML or 5'EML, was digested by SphI or KspI and resolved by PFGE. DNA transfer, hybridization, and image analysis were performed as described above. A repetitive sequence fingerprinting assay was performed as previously described (53, 54) with minor modifications. BAC or yeast DNA were digested by the 4-bp recognition restriction enzymes, HaeIII or HinfI, and electrophoretically resolved on 1% SeaKem agarose gels (FMC BioProducts) in 1x TAE running buffer. DNA was transferred to Nytran-Plus membranes (Schleicher & Schuell) by neutral transfer in 20x SSC. The probes were an equal-molar mixture of the oligonucleotides (GAA)₆, (GACA)₄, (GGGCA)₃, (GATA)₄, and (CT)₄(CA)₅. The probes were labeled by T4 polynucleotide kinase (NEB, Beverly, MA) following the manufacturer’s instructions. Membranes were prehybridized for 3 h and hybridized for 18 h at 42°C in 25 mM Tris, pH 7.5, 1 M NaCl, 50% formamide, 10% dextran sulfate, 1% SDS, 5x Denhart’s solution, with sonicated denatured salmon sperm DNA at 100 ng/ml. Wash conditions were 0.2x SSC, 0.1% SDS for 20 min at room temperature and twice for 30 min at 45°C. Membranes were exposed to PhosphorImage screens, and the images were analyzed using ImageQuant Software (Molecular Dynamics) as described above.

Fragmentation of yeast chromosome I

Because ML and 5'DML nearly comigrated with yeast chromosome I during PFGE, we bisected this yeast chromosome into 150-kb and 90-kb minichromosomes using a chromosome I fragmentation vector (55), creating a new yeast strain for propagation of these miniloci before their isolation. The bisection vector pFLC273 (a gift from Dr. David Kaback) was linearized with BamHI and transformed into yeast cells. Transformants obtained on selective medium for the URA3 marker were subsequently analyzed by PFGE.

Generation of transgenic mice carrying the Ig miniloci

Ig miniloci DNA was purified by PFGE according to standard protocols (56) with minor modifications. The YACs were separated by PFGE in 1% SeaPlaque agarose gels (FMC BioProducts), 0.5x TAE running buffer, with an auto algorithm set for a size range of 20–300 kb, using a Bio-Rad CHEF mapper apparatus. The gel slices corresponding to the YACs were excised. After equilibration in 1x TAE, slices were positioned on a gel tray and embedded in 4% Nusieve GTG agarose (FMC BioProducts). A second gel run was performed at a 90° angle to the PFGE for 3 h at 4 V/cm. The YAC-containing gel slices were excised and equilibrated in 1x TAE, 100 mM NaCl, 30 µM spermine, and 70 µM spermidine for 2 h at room temperature. The gel slices were melted at 68°C for 10 min and then treated with Gelase (Epicentre Technologies) at 1 U/100 mg of gel at 42°C for 2 h. The agarized solution was concentrated by centrifuging 30 min at 6000 x g in a Millipore filter unit, Ultrafree MC 30,000 NMWL (Millipore, Bedford, MA). The concentrated DNA was dialyzed on a Millipore type VM filter, pore size 0.05 µM, on the surface of microinjection buffer (10 mM Tris, 0.1 mM EDTA, 100 mM NaCl, 30 µM spermine, 70 µM spermidine) for 4 h at room temperature (56). The DNA was microinjected into C57BL/6 x SJL mouse zygotes, using 2 pl/cell at 0.5–3 ng/µl. Transgenic founders were identified by assay of tail DNA for the presence of left and right vector arms of the YAC constructs and were bred to C57BL/6 mice to create F₁ progeny for further analyses.

Southern hybridization analysis of mouse genomic DNA

Genomic DNA was isolated from mouse cells or tissues after incubation overnight at 55°C in 0.1 M EDTA, 0.5% N-lauroylsarcosine, 100 µg/ml proteinase K. Lysates were extracted once with equilibrated phenol:chloroform:isopropanol (25:24:1) and once with chloroform. DNA was precipitated by adding 2 vol of ethanol, briefly dried and resuspended in TE (10 mM Tris, 1 mM EDTA, pH 8.0). Samples were digested with restriction enzymes following the manufacturers’ recommendations and resolved in 1% agarose gels. DNA was transferred to Nytran-Plus membrane (Schleicher & Schuell) by neutral transfer in 20x SSC. Probes were labeled with [³²P]dCTP using the Rediprime II labeling system (Amersham) following the manufacturer’s instructions. Membranes were prehybridized for 2 h and hybridized to labeled probes for 18 h at 65°C as described above. Probed membranes were washed twice for 30 min in 0.2x SSC, 2% SDS and once for 30 min in 0.1x SSC, 2% SDS at 65°C. The images were analyzed using ImageQuant Software (Molecular Dynamics) as described above.

Analyses of copy numbers of Ig miniloci in transgenic mice

Copy numbers of both the 5' and 3' regions of Ig miniloci were determined by Southern analyses of mouse tail genomic DNA as described above. Because a point mutation was introduced into the miniloci that eliminated a BclI site in the C region, digestion of tail DNA with BglII/BclI and hybridization with a BglII/BclI probe isolated from plasmid pJC6.8 (34) yields a 2.4-kb band derived from the two endogenous alleles and a 2.8-kb band derived from the transgenes. Thus, the following equation was used to estimate copy number of the 3' region: 3' copy number = (signal of 2.8 kb/signal of 2.4 kb) x 2. The same filters were hybridized with the P5 probe amplified from the 5' region upstream of the most distal V2 gene segment as described above. The hybridization gives rise to a 1.1-kb band that is derived from both the transgenes and endogenous locus. Using the 1.1-kb band from a nontransgenic mouse tail DNA as the reference (copy number = 2) and the 2.4-kb C band as an internal loading normalization control, the 1.1-kb band from the transgenic mice tail DNA was quantified as that which represented the copy number of the 5' region as the sum of the transgenes and endogenous alleles. Subtraction of 2 gave rise to the estimated 5' copy number. The following equation was used for calculation: 5' copy number = (signal of transgenic 1.1 kb/signal of nontransgenic 1.1 kb) x 2 - 2. However, the 5' upstream region where the P5 probe was derived from had been deleted in the 5'DML. To circumvent this problem, the filter was hybridized to a neomycin resistance gene probe. Because all three miniloci contain a single copy of the neomycin resistance gene in the 5' vector arm, the 5' copy number of the transgene in the 5'DML mice was estimated by quantifying signals of hybridization with the neomycin probe using DNA with known 5' copy numbers from ML transgenic mice lines as the reference. For instance, hybridization of tail DNA isolated from transgenic mice 57-2 and 14-1 with the neomycin probe gave rise to bands with the same intensity after normalization. Thus, the 5' copy number in transgenic line 57-2 is estimated to be 3, the same as that of 14-1.

RT-PCR assay for germline transcription

The RT-PCR for germline transcription was performed as described previously (24). The primers were as follows (5' to 3'): ⁰, ACA GCC AGA CAG TGG AGT ACT ACC; C, TTA GTG GCT CTG TTC CTA TCA CTG TGT CCT CAG G. Reactions in 100 µl were performed with cDNA reverse transcribed from 100 ng total RNA isolated from mouse tissues using the Superscript System (Boehringer Mannheim, Mannheim, Germany) and 5 pmol of each primer at 3 mM Mg²⁺ using the Expand High Fidelity PCR System (Boehringer Mannheim) for 1 min at 94°C, 2 min at 60°C, 1 min at 72°C for 30 cycles. One picomole of the ⁰ primer labeled with [-³²P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) was added to the PCR products after 30 cycles. The final cycle was 1 min at 94°C, 2 min at 60°C, 10 min at 72°C. Then, 10 µl of the PCR products was digested with BclI and electrophoretically separated on 1.5% agarose gels in 1x TAE running buffer. Dried gels were exposed to PhosphorImaging screens, and images were analyzed as above.

PCR assay for V-J joining

PCR assay for gene rearrangement was performed based on the strategy developed by Schlissel and Baltimore (57). The PCR primers for the rearrangement assay were as follows (5' to 3'): V2 primer, GTC AAG TCA GAG CCT CTT AGA TAG TGG AAA GAC ATA TTT; V9 primer, GCA AGG CGA GTC AGG ACA TTA ATA GCT ATT TAA GCT GG; V20 primer, GTC ACT ATC AGA TGC ATA ACC AGC ACT GAT ATT GAT GAT GAT; V21 primer, GTG AAA GTG TTG AAT ATT ATG GCA CAA GTT TAA TGC AGT; J5 primer, GAG CCC TCT CCA TTT TCT CAA GAT TTT CTG AAC TG. Reactions in 100 µl were performed with 500 ng splenic DNA template and 5 pmol of each primer at 3 mM Mg²⁺ using the Expand High Fidelity PCR System (Boehringer Mannheim) for 1 min at 94°C, 2 min at 65°C, and 1 min 20 s at 72°C for 30 cycles. One picomole of the V2 primer labeled with [-³²P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) was added to the PCR products after 30 cycles. The final cycle was 1 min at 94°C, 2 min at 65°C, and 10 min at 72°C. Then, 10 µl of the PCR products were digested with the restriction endonucleases NcoI or AvrII and electrophoretically separated on 1% agarose gels in 1x TAE running buffer. Dried gels were analyzed as above.

Analyses of N regions

V2-J1 and V2-J2 rearrangements were amplified by PCR. The PCR primers used were the V2 primer described above and a J3 primer, CCT TTC TCA TTT CTC CCA CAA ATC TGA. The PCR conditions were 1 min at 94°C, 2 min at 60°C, 1 min at 72°C for 30 cycles using Tag DNA polymerase (Boehringer Mannheim). The PCR products were resolved on 1% agarose gel. The 1-kb V2-J1 and 700-bp V2-J2 rearrangement amplification products were gel-purified using Gel Extraction Kit (Qiagen, Chatsworth, CA) and subsequently cloned into the pGEM-T vector (Promega, Madison, WI). After sequencing, the rearrangement observed in individual clones could be deduced to correspond to either that of the endogenous alleles or transgenes, because of the presence or absence of AvrII/NcoI sites in the inserts.

RT-PCR assay for transcription of rearranged Ig genes

The primers for rearranged gene transcription were the V and C primers described above. Reactions in 100 µl were performed with cDNA reverse transcribed from 100 ng total RNA isolated from mouse tissues and 5 pmol of each primer at 3 mM Mg²⁺ using the Expand High Fidelity PCR System for 1 min at 94°C, 2 min at 65°C, 1 min at 72°C for 30 cycles. One picomole of the ⁰ primer or a V primer that was labeled with [-³²P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) was added to the PCR products after 30 cycles. The final cycle was 1 min at 94°C, 2 min at 65°C, 10 min at 72°C. Then, 10 µl of the PCR products were digested with BclI and electrophoretically separated on 1.5% agarose gels in 1x TAE running buffer. Gels were dried and analyzed as described above.

Hybridoma generation and analysis

Activated B cells from 4-day LPS-cultured splenocytes were fused to the non--producing myeloma cell line P3X63Ag8 (58) by standard techniques. Supernatants from clones growing in hypoxanthine/aminopterin/thymidine-supplemented RPMI 1640 medium were screened for Ig production by ELISA. Ninety-six-well plates were coated with 3 µg/ml salmon sperm DNA before ELISA. Cells in positive wells were then subcloned by limiting dilution. Total RNA was isolated from expanded clones, and Northern hybridization analysis was performed using 10 µg/lane as described elsewhere (59).

Isolation of Ig-expressing B cells

Single-cell suspensions were prepared from spleen and 4 x 10⁷ cells were incubated with an optimal concentration of biotinylated Abs in 100 µl PBS/0.1% BSA for 10 min on ice. After washing with PBS/0.1% BSA, cells were incubated with 20 ml of streptavidin microbeads (Miltenyi Biotech, Auburn, CA) for 30 min at 4°C. After removal of unbound microbeads, the cells were loaded into a mini-MACS unit (Miltenyi Biotech). Columns were washed three times with 200 µl PBS/0.1% BSA before the positive fraction was eluted following the manufacturer’s instructions. During the procedure, aliquots of fractions were stained with streptavidin-PE for FACS analysis to monitor the quality of the fraction. The fractionated cells were lysed with 0.5% N-lauroylsarcosine, 100 mM EDTA, 100 µg/ml proteinase K for genomic DNA isolation.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Structure of Ig miniloci

Using state-of-the-art techniques for manipulating YACs, we created a parent 240-kb Ig minilocus (ML) by recombining a 5' YAC (FAR.E8) with a 3' YAC (FAW.A3), separated by some 3 Mb in the Ig locus (1, 46, 52, 60 ; see Materials and Methods). As shown in Fig. 1, based on previous physical mapping studies (1, 18, 19, 23, 24, 25, 26), ML is predicted to contain all three functional members of the V2 gene family and one functional member of the V21 gene family, along with uninterrupted sequences spanning from V21G to the 3' RS. This predicted structure was verified by numerous assays (see below). To distinguish the gene rearrangement and expression of ML from those of the endogenous alleles by PCR and RT-PCR assays (see below), ML also contained engineered single base changes, converting an AvrII site between the J2 and J3 regions to an NcoI site and eliminating a BclI site in the C region by introducing a silent mutation. To test the hypothesis that the sequences 5' of the most distal V2 gene family member, V2(70/3), may provide regulatory functions to downstream V2 genes, we also created deleted and extended forms of ML, termed 5'DML and 5'EML, respectively (Fig. 1). In summary, the YAC-based engineering lead to the creation of three miniloci with differing lengths of sequences upstream of the V2 gene cluster (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Structure of Ig miniloci. Shown are ML, 5'DML, and 5'EML, which possess 15 kb, 0.1 kb, and 65 kb, respectively, of sequence upstream of the most 5' V2 gene. The physical map of V regions along with their indicated transcriptional orientation is from our results and the extensive studies of Zachau and coworkers (1 18 19 23 24 25 26 61 ). Not shown are the positions of nonfunctional V2, V9, and V20 pseudogenes (23 24 25 26 ). Shown below are the three parent DNA constructs that were fused together to create 5'EML. The precise position of the breakpoint fusion lies somewhere within the bracketed region between FAR.E8 and FAW.A3. Not to scale are the YAC vector arms, which possess telomeres, a centromere (CEN), an origin of replication (ARS), along with the indicated selectable markers.

Validation of Ig miniloci

To confirm that ML originated from recombination between YACs FAR.E8 and FAW.A3d and roughly map the break points, we conducted an indirect end-labeling assay (Fig. 2A). DNA from the two parent YACs and ML was partially digested with SfiI; after PFGE, Southern transfers were hybridized to vector arm probes from each parent YAC. Comparison of the resulting banding patterns between FAR.E8 and ML revealed a break point located between 165 and 215 kb downstream of the 5' end of FAR.E8 (Fig. 2A, left, broken arrow). Similarly, the junction was identified between 70 and 90 kb upstream of the 3' end of FAW.A3d (Fig. 2A, right, broken arrow). The sum of these breakpoint values is within the range of the length of ML as analyzed by PFGE, indicating that no significant internal deletion occurred during recombination. Southern analyses and PCR assays has revealed that in addition to the J-C region, the ML contains the expected V2 and V21 gene family members (Ref. 61 and data not shown). Junctions between the ML and the 5'EML were also investigated by Southern analyses (Fig. 2B). A 40-kb SphI fragment in 5'BAC was retained in 5'EML as well as an 85-kb KspI fragment (Fig. 2B, 1–3, arrows). The 8-kb vector at the 5' end of 5'EML accounts for the length discrepancy between the KspI fragments of the 5'BAC and 5'EML, and the top band in the KspI-digested BAC resulted from partial cleavage due to inhibition by bacterial specific methylation (Fig. 2B, 2). Furthermore, the extension of the 5' region was confirmed by genomic fingerprinting assays. ML, 5'EML, and 5'BAC DNA digests were hybridized to mouse genome simple repeat probes. 5'EML digests contained all bands present in the ML digests and those bands that were unique in the BAC digests (Fig. 2B, 4 and 5, arrows), consistent with the lack of deletions or aberrant rearrangements. Finally, Southern hybridization analysis of the products of chromosome fragmentation at of ML at V2 gene segments indicated that ML contains 15 kb of upstream sequence (Fig. 2C, compare the length of ML in lane 1 to those 5'DML fragmentation products in lanes 3–4, 6, and 8, arrow). We conclude that the Ig miniloci thus generated behave in all ways tested predictable for their anticipated structures.

View larger version (80K): [in this window] [in a new window]   FIGURE 2. Southern hybridization analysis of Ig miniloci. A, Indirect end-labeling assay to deduce junctions in the ML. FAW.A3d represents fragmented FAW.A3 in which chimeric material was eliminated (see Materials and Methods). In the 5' junction assay, electrophoretically resolved SfiI partially digested DNA was hybridized to the R probe. In the 3' junction assay, electrophoretically resolved SfiI partially digested DNA was hybridized to the L probe. The approximate positions for break points are indicated by the broken arrows. B, Analyses of the structure of the 5'EML by Southern hybridization. 1–3, Southern hybridization of SphI- or KspI-digested 5'BAC, ML, and 5'EML DNA with the indicated probes. The doublet KspI-digested 5'BAC bands are due to inhibition of complete cleavage by bacterial-specific methylation. 4 and 5, Fingerprinting assays using repeated sequences as probes. Arrows indicate fragments in both the 5'BAC and 5'EML that are not found in ML. C, Southern hybridization analysis of products resulting from fragmentation of ML at V2 gene segments to identify 5'DML and to map the length of sequences upstream of the 5' V2 gene segment. Lane 1, ML; lanes 2–8, ML fragmentation products. Note that the shorter products in lanes 2, 5, and 7 support the assigned position of the middle V2 gene segment in the physical map shown in Fig. 1.

Generating transgenic mice carrying Ig miniloci

We isolated the three Ig miniloci constructs described above by PFGE for microinjection into fertilized mouse eggs to create transgenic mice. A total of 16 different transgenic mice were identified by dot blot hybridization using miniloci vector arm probes, 14 of which transmitted the transgenes to F₁ progeny (Table I). The copy numbers of transgenes was determined by genomic Southern hybridizations using 5' and 3' end probes. A representative Southern assay for the 3' copy number estimation in founder animals is shown in Fig. 3. Because a point mutation was introduced into the miniloci that eliminated a BclI site in the C region, after digestion of tail DNA with BglII/BclI, the corresponding endogenous and transgene fragments are 2.4 and 2.8 kb, respectively (Fig. 3), the relative signal intensities of which were used for quantitation. Estimate of 5' copy number used band signal intensities relative to internal and external standards (Ref. 61 and data not shown). The copy numbers of transgenes was found to range from 1 to 14 (Table I). Among the16 transgenic mouse lines, 11 had the same copy numbers for both 5' and 3' ends of the construct, suggesting that most integrated miniloci were intact, possibly organized as tandem arrays (62). It can also be deduced that at least one copy of these constructs must be intact in every transgenic line created from the fact that all such lines exhibited rearrangement of V2 and V21 genes (see below).

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Analysis of copy number of the 3' C region in Ig miniloci transgenes. A C region probe was hybridzed to BglII/BclI-digested genomic DNA isolated from wild-type (WT) or the indicated transgenic founders. The signal of the 2.4-kb band represents two copies of the endogenous Ig alleles. The 2.8-kb band represents transgenes.

Germline transcription of Ig miniloci is generally position independent and copy number dependent

To study Ig miniloci expression, we first evaluated germline transcription in F₁ progeny. By performing RT-PCR with spleen and bone marrow RNA samples, we assayed for the level of spliced germline transcripts produced from the 3' germline promoter (27). Taking advantage of the eliminated BclI site in the C region of the miniloci constructs, germline transcripts arising from the endogenous and transgene alleles could be distinguished by gel electrophoresis after BclI digestion of the RT-PCR products (Fig. 4, A and B). Fig. 4B illustrates a representative assay performed on wild-type and two F₁ transgenic mice progeny. Our quantitated results demonstrate that the levels of germline transcription in transgenes are approximately the same as those of the endogenous locus and exhibit position-independent and copy number-dependent expression (Fig. 4C), except for those transgenic lines with very high copy numbers.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Analysis of germline transcription of Ig miniloci. A, Scheme for RT-PCR analysis of germline transcription. 0 arrow, 3' germline promoter. A BclI site in the C region was eliminated in transgenes by a single base change resulting in a silent mutation (X). PCR amplification gives rise to 388-bp fragments that are 32P-labeled at their 5' ends. After digestion with BclI, the resistant 388-bp fragments represent transgene (T) products, while the resulting 233-bp fragments are derived from the endogenous (E) alleles. (Because a portion of unamplified samples form heteroduplexes where only one strand possesses the BclI site and would resist restriction digestion, we developed a strategy in which a 32P-labeled 5' primer was added before the last round of amplification and PCR products were analyzed by PhosphorImaging (81 ). This approach allows exclusive quantification of homoduplexes in the radioactive extended products.) B, Gel electrophoresis of PCR products before (-) and after (+) BclI digestion. RT-PCR products were amplified from spleen (S) and bone marrow (B) total RNA samples of wild-type (WT) and two transgenic mice lines, 26-2 and 32-2. C, Quantification of the RT-PCR products shown in B, as well as data similarly generated from the other 14 transgenic lines shown in Table I. Error bars for each point represent the SD of the mean (n = 3). Insets, Summary of linear regression analyses (excluding data from mice with 8 and 14 copy numbers), with n equal to the total number of points; b the regression coefficient, or the slope of the best fit regression line; Sb the SE of the regression coefficient; and r the correlation coefficient.

V-J rearrangement of Ig miniloci approches endogenous levels for shared V regions and exhibits correct developmental timing and tissue specificity

To investigate the level of rearrangement of Ig miniloci, we assayed for V-J joining using a PCR assay (57). Specific primers for V2 and V21 families present in the miniloci were used to assay for their corresponding rearrangement. Four PCR products are expected that represent VJ1, VJ2, VJ4, and VJ5 rearrangements (Fig. 5A). Taking advantage of the converted AvrII site in the J region of the miniloci constructs, rearrangements arising from the endogenous and transgene alleles could be distinguished by gel electrophoresis after AvrII or NcoI digestion of the PCR products (Fig. 5, A and B). Fig. 5B illustrates a representative gel in which V2 rearrangement was analyzed from spleen and bone marrow total DNA samples of wild-type and two F₁ transgenic mice progeny. These and other signals from the transgenic mice were quantified and normalized to the level of rearrangement of the corresponding V region of the endogenous Ig locus (Fig. 5C). This analysis revealed that both V2 and V21 family members in transgenes were rearranged quite efficiently, exhibiting levels often equal to or greater than those exhibited by their endogenous counterparts (Fig. 5C). In addition, V2 but not V21 genes in 5'EML rearranged at a greater level than those in 5'DML and ML constructs in most transgenic animals (p < 0.005).

View larger version (56K): [in this window] [in a new window]   FIGURE 5. PCR assay for V-J rearrangement. A, Scheme for the PCR assay. A 5' primer for a specific V family and a universal 3' primer are used for amplification. Last-round extension with a radioactively labeled primer was conducted to overcome heteroduplex problems as described in Fig. 4. In Ig miniloci, the AvrII site between J2 and J3 was converted to an NcoI site. The indicated four rearrangement products are produced by amplification. V-J1 and V-J2 products can be digested with AvrII or NcoI to generate two truncated fragments labeled at their 5' ends. B, Gel electrophoresis of the PCR products. Shown in the gel are V2 gene amplified rearrangement products from spleen or bone marrow DNA samples of a wild-type (WT) and two transgenic mice, 19-5-1 and 26-2. Four products and two digested 5' fragments (VJ1–5' and VJ2–5') are indicated. C, Summary of V-J1 rearrangement levels from V2 and V21 genes in the Ig miniloci. The levels of rearrangement are represented as fold of those from the same V family of the endogenous alleles. We have not normalized for transgene copy number on the assumption that miniloci obey allelic exclusion. The numbers in the x-axis indicate transgenic lines. For the founder animal 14-1 and F1 mice bearing the 5'DML, the PCR assay was repeated three times using spleen and bone marrow DNA isolated from the single animals. For each other transgenic line, the PCR assay was repeated using DNA samples isolated from three different F1 mice. S, Spleen; B, bone marrow. Error bars represent the SD of the mean (n = 3). Similar results were obtained for the level of rearrangement of V24 in 5'EML (data not shown).

View this table: [in this window] [in a new window]   Table II. Nucleotide sequences at V2-J1 junctions of miniloci1

View larger version (50K): [in this window] [in a new window]   FIGURE 6. Ig miniloci are not rearranged in T cells. Summary of V2-J1 rearrangement levels from the indicated Ig miniloci represented as fold of those of the endogenous alleles. We have not normalized for transgene copy number on the assumption that miniloci obey allelic exclusion. The numbers in the x-axis indicate transgenic lines. Error bars represent the SD of the mean (n = 3). Because endogenous alleles are not rearranged in T cells, the observation that the ratio of rearrangement of transgenes/endogenous is the same in T cells and B cells indicates that the trace of rearrangement detected is due to B cell contamination in the thymus tissue.

Transcription of rearranged Ig miniloci approaches endogenous gene levels

We also determined the level of transcription from rearranged transgenes by a RT-PCR assay using V2 and V21 primers and a C primer to amplify spliced transcripts (Fig. 7A). RT-PCR products resisting digestion by BclI represent transcripts derived from rearranged miniloci. Fig. 7B illustrates a representative gel in which cDNAs arising from rearranged V2 genes were analyzed from spleen and bone marrow total RNA samples of wild-type and two F₁ transgenic mice progeny. These and other transgenic mice signals were quantified and normalized to the level of transcription of the corresponding rearranged V region of the endogenous Ig locus, as well as for their corresponding level of relative rearrangement (Fig. 7C). In most F₁ transgenic mice, the level of transcription of the rearranged miniloci approached those of endogenous loci, with rearranged V2 and V21 of the miniloci transcribed at an average of 0.5- and 0.8-fold the level of corresponding endogenous counterparts. In addition, the variation in Ig miniloci transcription levels between different transgenic lines indicated position effects. Interestingly, V2 and V21 of the miniloci in the same transgenic animals exhibited different sensitivities to position effects, for both the level of rearrangement (Fig. 5) and the level of transcription of rearranged genes (Fig. 7), suggesting that V2 and V21 chromatin accessibility for rearrangement and promoters for rearranged gene transcription are intrinsically different in responding to repressive effects of neighboring chromosomal environments. Despite the difference in the level of rearrangement, transcription of rearranged V2 transgenes exhibited little difference between the three miniloci (p > 0.1). Similarly, the level of mRNA transcribed from rearranged V21 transgenes was not distinguishable between the three constructs (p > 0.1).

View larger version (63K): [in this window] [in a new window]   FIGURE 7. RT-PCR analysis of transcription from rearranged Ig genes. A, Scheme for the RT-PCR assay. A 5' primer for a specific V family and a universal C 3' primer are used for amplification. Last-round extension with radioactively labeled primers was conducted to overcome heteroduplex problems as described in Fig. 4 (81 ). The BclI site in the C-coding region was eliminated in transgenes by a single base change resulting in a silent mutation (X). B, Gel electrophoresis of the PCR products. Shown on the gel are V2-amplified RT-PCR products from spleen or bone marrow RNA samples of a wild-type (WT) and two F1 transgenic mice, 19-5-1 and 26-2. After digestion with BclI, the resistant 588-bp fragments represents transgene (T) products, while the resulting 433-bp fragments are derived from the endogenous (E) alleles. C, Quantification of the RT-PCR assay. The level of transcription is represented as fold of those from the same V family of the endogenous alleles, normalized for their relative extent of rearrangement. The numbers on the x-axis indicate transgenic lines. S, Spleen; B, bone marrow. Error bars represent the SD from the mean (n = 3). Similar results were obtained for the level of transcription of rearranged V24 in 5'EML (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 8. Northern analysis of Ig mRNA levels in hybridomas generated from transgenic line 19-5-1 expressing functionally rearranged V2 genes of the minilocus (lanes 3–8) or endogenous Ig alleles (lanes 9–14).

Evidence that Ig miniloci obey allelic exclusion

Previous transgenic experiments using germline human Ig constructs containing a single V gene segment showed very high levels of rearrangement with essentially every multiple copy of the transgenes exhibiting rearrangement in every B cell (40). Thus, these transgenes did not obey allelic exclusion. In contrast, the lower level of rearrangement exhibited by our Ig miniloci is consistent with the possibility that their rearrangement may be excluded by the endogenous Ig locus or other copies of the transgenes. To more directly evaluate allele usage for productive V-J joining in transgenic mice, we isolated Ig-producing B lymphocytes from two selected transgenic lines using anti-Ig biotinylated Abs and streptavidin-coated magnetic beads. FACS analysis revealed that the Ig⁺ cell population was >95% pure (Fig. 9A). To evaluate allele usage in the Ig⁺ cell population, we performed Southern analysis to specifically detect the unrearranged 1.2-kb DraI/AvrII or 2.6-kb DraI/NcoI fragments of the endogenous allele (E) and the corresponding 3.6-kb DraI or 1.2-kb DraI/NcoI fragments of the transgene (T) (Fig. 9B). Miniloci were less used for rearrangement than the endogenous alleles, because a higher percentage of transgenes existed in germline configuration in Ig⁺ cell DNA compared with tail DNA (Fig. 9C, e.g., compare lanes 5 and 6 with 7 and 8). These results provide evidence that our Ig miniloci are responsive to allelic exclusion and prove that not all copies of our transgenes become rearranged in B cells.

View larger version (51K): [in this window] [in a new window]   FIGURE 9. The usage of Ig miniloci and endogenous alleles in trangenic mice. A, Ig-producing B cells were isolated from splenic cell suspensions with biotinylated anti-Ig in combination with avidin-magnetic beads in mini-MACS columns. The purity of isolated cells was determined by FACS. Input, Total population of splenic cells. Unbound, The negative fraction not bound to the mini-MACS column. Bound, The eluted positive fraction. B, Physical map of the J region. The probe is indicated by the bar. Also indicated are the lengths of key restriction fragments. C, Southern blot using genomic DNA derived from tails or isolated Ig producing B cells. T, Transgene germline bands; E, endogenous germline bands.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of Ig miniloci using YAC-based technology

Owing to the availability of a variety of forward- and reverse-genetic tools in yeast, YACs are a powerful resource for engineering large DNA constructs. Modifications can be as dramatic as large deletions or as subtle as single base changes. For example, we modified two parent YACs by means of gene replacement, chromosome fragmentation, and introduced single base changes in the minilocus. Given the observation that chimerism can approach 70% during the generation of YAC libraries due to a high frequency of recombination between cotransformed DNA fragments (70), we developed a novel strategy to create recombinant YACs (see Materials and Methods). In comparison to meiotic and mitotic recombination approaches, this method is less time-consuming and more efficient, especially for fusing YACs through heterologous sequences. Finally, we reasoned that an ideal Ig minilocus should contain sequences always preserved in the native locus after V-J joining and should not exceed several hundred kilobases for ease of introduction into transgenic mice or cultured cells. As summarized in Table IV and discussed in detail below, our analysis of various parameters of transgene expression is the most complete yet reported in the literature, and the results are quite rewarding with respect to the levels of expression achieved.

View this table: [in this window] [in a new window]   Table IV. Summary of Ig germline transgene studies

Germline transcription from the miniloci was nearly proportional to transgene copy number in both bone marrow and spleen tissue from transgenic animals, being similar in level per locus copy to that of the endogenous alleles, except for mice carrying high copy (8 or 14) transgenes, which exhibited reduced levels of germline transcription (Fig. 4C). Repeat-induced gene silencing has been observed in previous studies, which is possibly caused by a preference for integration into, or formation of, heterochromatin (71). Nevertheless, for the lower copy number transgenes, we can conclude that Ig miniloci contain all the necessary regulatory elements to specify position-independent and copy number-dependent germline transcription at a level equivalent to that of the endogenous locus. In contrast, germline transcription of much shorter transgenes has been reported to be sensitive to position effects (72). Our miniloci contained 200–250 kb of sequence 5' of the germline promoters and 25 kb of downstream sequence, including the two known enhancers and a MAR. It is possible that the lack of position effect is simply due to presence of these long stretches of DNA sequences that protect the germline promoters from the flanking chromosomal environment. However, transcription of rearranged V21 genes exhibited a substantial variation between transgenic lines despite the fact that both upstream and downstream sequences remain in rearranged V21 transgenes.

Position-dependent, copy number-independent rearrangement

Transgene rearrangement exhibited significant position effects, suggesting that the miniloci lack insulator or chromosomal domain boundary sequences that would override adverse flanking chromosomal environments (73, 74). The observations that rearrangement but not germline transcription exhibited position effects indicates that these processes are not necessarily coupled. This is consistent with other results that have disconnected these events (11), suggesting that germline transcription per se is not sufficient to direct efficient rearrangement. Alternatively, chromatin accessibility may be regulated by mechanisms in addition to or other than germline transcription, and transcription directed from germline promoters may simply be a consequence of an open chromatin structure. In addition, transgene rearrangement was not proportional to locus copy number, which may be in part due to allelic exclusion.

Our results reveal that the 5' and 3' V region genes, V2 and V21, are rearranged quite efficiently, up to several fold higher than their endogenous counterparts (Fig. 5C). This rearrangement was tissue specific as it did not occur in T cells (Fig. 6). In addition, lack of N regions at V-J junctions suggests that transgenes were rearranged at the proper stage in B cell development (Table II). Interestingly, V2 but not V21 genes in 5'EML rearrange more efficiently than those in 5'DML and ML, suggesting that rearrangement enhancing sequences may reside within the additional 50-kb upstream region in 5'EML (Fig. 5C).

At least two models can be postulated to explain the regulation of Ig locus rearrangement, assuming that allelic exclusion is equally obeyed for endogenous and transgene rearrangements. One model hypothesizes that a master regulator is present in the Ig locus that specifies locus commitment to rearrangement. If the probability for commitment to rearrangement of a given locus is determined by a master regulator and that regulator is present in our miniloci, then the miniloci would have the same probability per locus copy for rearrangement as that of the endogenous alleles. There are only four functional V genes in ML in comparison to 93 functional V genes in an endogenous allele (23, 24, 25). Thus, the master regulator model would roughly predict per allele that a certain V gene segment in the miniloci would have 93/4 or a 23-fold greater chance for rearrangement than the endogenous counterpart, assuming all V regions exhibit an equal probability for rearrangement (for discussion purposes only). Alternatively, another model proposes that each V gene determines its own rearrangement potential. In this case, the more V regions that an Ig locus contains, the greater the probability that this locus would exhibit rearrangement during B cell differentiation. This "V gene number" theory is consistent with the observed / ratio in the mouse (20:1) and human (6:4). If V gene number determines the probability for locus rearrangement, then each endogenous locus would have 93/4 or a 23-fold greater chance for rearrangement relative to our transgene, assuming all V regions exhibit an equal probability for rearrangement (for discussion purposes only). A certain V gene segment in the miniloci would have the same chance for rearrangement as compared with the endogenous counterpart. PCR rearrangement data strongly support the "V gene number" model because the same V genes in both transgenes and the endogenous loci exhibited very similar levels of rearrangement in most cases, except V2 in 5'EML (Fig. 5C). In addition, genomic Southern analyses of Ig alleles in -producing splenic cells reveals that <10% of the transgenes had undergone rearrangement (limits of sensitivity) (Fig. 9), consistent with their responsiveness to allelic exclusion. Previous transgenic experiments using germline human Ig constructs containing a single V gene segment showed very high levels of rearrangement, supporting the master regulator model. However, these transgenes did not obey allelic exclusion and therefore were aberrantly expressed (40). It is also possible that locus activation is determined not only by the number of V gene segments but also by the strength of their promoters for specifying germline transcription (75, 76, 77, 78).

Analysis of transcription from rearranged transgenes

Previous studies employing much shorter rabbit or human germline Ig constructs containing the intronic enhancer have observed a dramatic disproportionality between the levels of rearrangement and transcription (40, 67, 72). In our studies, rearranged V2 and V21 regions of the miniloci were transcribed at levels approaching those of their endogenous gene counterparts, although they still exhibited strong position effects. Northern analysis of rearranged V2-expressing hybridomas generated from transgenic mice also revealed high-level transcription of rearranged miniloci. It is significant that this relatively efficient level of transcription has never been observed in previous transgenic studies employing shorter germline Ig gene constructs (Table IV), indicating that three Ig miniloci analyzed in our studies contain essentially all regulatory elements to confer copy number-dependent, position-independent germline transcription, tissue and developmental stage-specific efficient V-J rearrangement, and rearranged Ig gene transcription. These features make these miniloci useful reagents for future functional analyses of regulatory elements in the locus. Although 5'EML and 5'DML differ by having either 65 kb or only 0.1 kb of sequence upstream of the 5' V2 gene, it is surprising that the only difference observed between these constructs was the level of recombination of the V2 gene segments. Finally, because the Ig miniloci still exhibit position effects, it is possible that they are missing domain boundary elements and a locus control region (73, 74, 79, 80). Nevertheless, our analysis places limits on where these hypothetical elements might be found, within a 15-kb region between the RS and the downstream ribose-5-phosphate isomerase gene (49), and/or >65 kb upstream of V2 (70/3) gene.

	Footnotes

 
¹ This investigation was supported by Grants GM29935 and GM51585 from the National Institutes of Health and Grant I-823 from the Robert A. Welch Foundation (to W.T.G.).

² Address correspondence and reprint requests to Dr. William T. Garrard, Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9148. E-mail address:

³ J. B. George-Raizen, Si Li, and W. T. Garrard. A pre-B cell specific silencer in the mouse Ig gene locus. Submitted for publication.

⁴ Abbreviations used in this paper: MAR, nuclear matrix association region; ML, Ig gene minilocus; 5'DML, 5'-deleted ML; 5'E ML, 5'-extended ML; PFGE, pulsed-field gel electrophoresis; RS, recombination sequence; YAC, yeast artificial chromosome.

Received for publication June 22, 1999. Accepted for publication October 26, 1999.

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