metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus ( tcr c-17317 and tcr v-rp24 - 74e19 ) , the igh locus ( igh v-224m14 ) , igh c ( from c1- 3of c ) and a telomere - repeat specific peptide nucleic acid ( pna ) probe ( applied biosystems ) .
three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described .
confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat ( n.a .
1.4 ) objective lens with 0.07 m x - y pixel sampling , optical slice thickness of 0.8 m , and z - step size of 0.2 m .
volumes of interest ( vois ) were drawn around fish spots to be measured . within the individual vois ,
the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software ( mipav , cit / nih ) .
tetraspeck fluorescent microspheres ( molecular probes / invitrogen ) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope , fluorescence channel alignment , and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object .
the minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . for distance measurements
the following tcr probe sets were used : rp24 - 334b8 and rp23 - 255n13 ( proximal ) , rp24 - 74e19 and 17317 ( middle ) , rp23 - 304l21 and rp23 - 10k20 ( distal ) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d .
thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4 , cd8 , tcr ( h57 - 597 ) , tcr  ( gl3 ) ( bd pharmingen , san diego , ca ) .
genomic dna was isolated from thymocytes using a qiagen dna isolation kit ( qiagen , valencia , ca ) .
50ng dna was pcr amplified with a combination of gene - specific primers ( supplementary table 3 ) using platinum taq polymerase ( invitrogen , carlsbad , ca ) for standard , or the power sybr green master mix kit ( applied biosystems , foster city , ca ) for qpcr .
real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software ( applied biosystems , foster city , ca ) . for each assay , aliquots of dna were analyzed for a control , non - rearranging dna 3 of j2 .
the cycle threshold numbers for each primer combinations ( ct ) and for the control amplification ( ct ) were used to calculate the absolute amount of pcr signal .
the relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . for sequence analysis ,
150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit ( invitrogen ) .
amplify tcr v - dj coding joint sequences , v primers were used as forward primers with the primer 3j1 as reverse primer ( supplementary table 4 ) .
junctions from tcr mice were amplified using the indicated primers ( supplementary table 4 ) .
briefly , 10 g of total thymus genomic dna was digested with stui ( new england biolabs ) .
the digest products were run out on a 1% agarose tae gel , transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends .
one g of genomic dna was treated with terminal deoxynucleotidyl transferase ( new england biolabs ) per manufacturer s protocol at a final concentration of 5 m datp .
two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers .
conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute , 57c for 45 seconds , and 72c for 45 seconds and then a final 5 minute extension step at 72c .
two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water .
one l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii .
reaction conditions were the same as for the primary reactions , but the total number of cycles was increased to 30 cycles total .
15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane ( bio - rad ) .
loading control pcr reactions , aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described .
10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane ( bio - rad ) .
the oligonucleotides d1 , d2 , univ - bglii , t17-univ and ds850 are listed in supplementary table 4 .
thymi were fixed in buffered 10% formalin , and paraffin sections were stained with hematoxylin - eosin .
metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus ( tcr c-17317 and tcr v-rp24 - 74e19 ) , the igh locus ( igh v-224m14 ) , igh c ( from c1- 3of c ) and a telomere - repeat specific peptide nucleic acid ( pna ) probe ( applied biosystems ) .
three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described .
confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat ( n.a .
1.4 ) objective lens with 0.07 m x - y pixel sampling , optical slice thickness of 0.8 m , and z - step size of 0.2 m .
volumes of interest ( vois ) were drawn around fish spots to be measured . within the individual vois ,
the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software ( mipav , cit / nih ) .
tetraspeck fluorescent microspheres ( molecular probes / invitrogen ) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope , fluorescence channel alignment , and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object .
the minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . for distance measurements
the following tcr probe sets were used : rp24 - 334b8 and rp23 - 255n13 ( proximal ) , rp24 - 74e19 and 17317 ( middle ) , rp23 - 304l21 and rp23 - 10k20 ( distal ) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d .
thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4 , cd8 , tcr ( h57 - 597 ) , tcr  ( gl3 ) ( bd pharmingen , san diego , ca ) .
genomic dna was isolated from thymocytes using a qiagen dna isolation kit ( qiagen , valencia , ca ) .
50ng dna was pcr amplified with a combination of gene - specific primers ( supplementary table 3 ) using platinum taq polymerase ( invitrogen , carlsbad , ca ) for standard , or the power sybr green master mix kit ( applied biosystems , foster city , ca ) for qpcr .
real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software ( applied biosystems , foster city , ca ) . for each assay , aliquots of dna were analyzed for a control , non - rearranging dna 3 of j2 .
the cycle threshold numbers for each primer combinations ( ct ) and for the control amplification ( ct ) were used to calculate the absolute amount of pcr signal .
the relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . for sequence analysis ,
150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit ( invitrogen ) .
amplify tcr v - dj coding joint sequences , v primers were used as forward primers with the primer 3j1 as reverse primer ( supplementary table 4 ) .
junctions from tcr mice were amplified using the indicated primers ( supplementary table 4 ) .
briefly , 10 g of total thymus genomic dna was digested with stui ( new england biolabs ) .
the digest products were run out on a 1% agarose tae gel , transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends .
one g of genomic dna was treated with terminal deoxynucleotidyl transferase ( new england biolabs ) per manufacturer s protocol at a final concentration of 5 m datp .
two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers .
conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute , 57c for 45 seconds , and 72c for 45 seconds and then a final 5 minute extension step at 72c .
two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water .
one l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii .
reaction conditions were the same as for the primary reactions , but the total number of cycles was increased to 30 cycles total .
15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane ( bio - rad ) .
the membrane was subsequently hybridized with p - labeled ds850 oligonucleotide . for il-2 loading control pcr reactions , aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described .
10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane ( bio - rad ) .
the oligonucleotides d1 , d2 , univ - bglii , t17-univ and ds850 are listed in supplementary table 4 .
thymi were fixed in buffered 10% formalin , and paraffin sections were stained with hematoxylin - eosin .
 v(d)j recombination and class switch recombination employ overlapping but distinct non - homologous end - joining ( nhej ) pathways to repair dna double strand break ( dsb ) intermediates . 
 53bp1 is a dna damage response protein that is rapidly recruited to sites of chromosomal dsbs , where it appears to function in a subset of ataxia - telangiectasia mutated ( atm ) kinase , h2ax- and mdc1- dependent events1,2 . 
 a 53bp1 dependent end joining pathway has been described that is dispensable for v(d)j recombination but essential for class - switch recombination csr3 , 4 . here , we report a previously unrecognized defect in the joining phase of v(d)j recombination in 53bp1 deficient lymphocytes distinct from that found in classical nhej- , h2ax- , mdc1- and atm - deficient mice . 
 absence of 53bp1 leads to impairment of distal v - dj joining with extensive degradation of un - repaired coding ends and episomal signal joint reintegration at v(d)j junctions . 
 this results in apoptosis , loss of t - cell receptor alpha locus integrity and lymphopenia . 
 further impairment of the apoptotic checkpoint causes propagation of lymphocytes bearing antigen receptor breaks . 
 these data suggest a more general role for 53bp1 in maintaining genomic stability during long range joining of dna breaks . 