Supplementary Information

Transcription

1 Supplementary Information Supplementary Figure 1! a! b! Nfatc1!! Nfatc1"! P1! P2! pa1! pa2! ex1! ex2! exons 3-9! ex1! ex11!!" #" Nfatc1A!!" Nfatc1B! #"!" Nfatc1C! #" DN1! DN2! DN1!!A! #A!!B! #B!!C! #C!!A! #A!!B! #B!!C! #C! DN1! DN2! Actb! Nfatc1! 38 cycles! 45 cycles! c! d! DN ! DN4! NFATc1!" Nfatc1!! DN3! DN4! Nfatc1"! Actb! DAPI! 1

2 Supplementary Figure Legends Supplementary Figure 1 Exclusive Nfatc1 P2 promoter activity in ptcr-negative thymocytes (a) Schematic representation of the Nfatc1 gene and the six Nfatc1 isoforms derived from P1 and P2 promoters due to alternate splicing and usage of two different polyadenylation (pa) sites. (b) RT-PCR analysis of Nfatc1 isoforms expression in sorted WT DN1 and DN2 cells. Only Nfac1β isoforms derived from the P2 promoter was detectable even with increased number of PCR cycles. (c) Analysis of total P1 or P2 promoter-derived Nfatc1 transcripts in freshly isolated WT DN3 and DN4 cells. (d) Immunofluorescence analysis of NFATc1α levels in sorted WT ptcr-negative (DN1+2+3) and -positive (DN4) cells. Nuclear NFATc1α was confirmed with counter staining with DAPI. Scale bar, 1 µm. 2

3 Supplementary Figure 2! a! E! X! H! C! T!X! V! E! ex.1! ex.2! ~ 14.7kb! ~ 6.5kb! WT Nfatc1! E! X! H! E! V! T!X! V! E! ex.1! ex.2! Nfatc1 P2 targeting! PGKneo! construct! ~ 6.8kb! ~ 8.3kb! b! E! X! H! T!X! V! E! ex.1! ex.2! ~ 12.2kb! 21.4 Kb! 6.8 Kb! c! d! Nfatc1 P2 targeted! construct!.floxed P2! allele (8 Kb)! P2 flox! ( bp)! P2 WT! (365 bp)! e! ! P2 f/f! 41! Vav-CreP2 fl/+! Vav-CreP2 fl/fl! 43! f! P2 fl/fl! Vav-CreP2 fl/fl! 1 2 CD4! 1 1 3! 26! 25! CD8! Cells! Isotype! ic Bcl-2! 3

4 Supplementary Figure 2 Normal T cell development in Nfatc1 P2 promoter-deficient mice (a) Gene targeting strategies to generate the targeting vector for generation of the Nfatc1 P2 promoter floxed mice. Rectangles and triangles represent exon and flox sites respectively. Restriction enzyme sites are denoted in capital letters; EcoRI (E), XbaI (X), XhoI (H), Eco72I (C), AatII (T), EcoRV (V). (b) Southern blot to detect embryonic stem (ES) cell clones positive for the targeted P2 floxed allele. (c) Long distance PCR with genomic tail DNA to detect the integration of the floxed P2 targeting vector. (d) Genotyping PCR to detect WT (365 bp), heterozygous and homozygous ( bp) P2 floxed mice using genomic tail DNA. (e) CD4 + and CD8 + T cells distribution in lymph nodes from WT, Vav-CreNfatc1P2 fl/+ and Vav-CreNfatc1P2 fl/fl mice. Numbers inside each plot represent percent respective population. (f) Intracellular Bcl-2 levels in DN3 cells from WT (P2 fl/fl ) and Vav-CreNfatc1P2 fl/fl mice. Data are representative of 3 independent experiments (e & f), (n = 3 per group per experiment). 4

5 Supplementary Figure 3! a! % cells!!!!!!! Nfatc1!" fl/fl! Vav-CreNfatc1!" fl/+! Vav-CreNfatc1!" fl/fl! ***! **! DN3! DN4! b! % of Max ic CD3!" Isotype! DN3 cells! WT (MFI 4)! Vav-CreNfatc1!" fl/fl (MFI 41)! c! 3! Thymus! 25! Cell number (x1 6 )!! 15!! WT (n = 9)! Vav-CreNfatc1αΑ fl/fl (n = 7)! Vav-CreNfatc1 fl/fl (n = 9)! Vav-CreNfatc1αΑ fl/fl Nfatc1 fl/fl (n = 3)! 5!! d! WT! Vav-CreNfatc1αΑ fl/fl! Vav-CreNfatc1 fl/fl! Vav-CreNfatc1αΑ fl/fl Nfatc1 fl/fl! 49! 2! 79! 4!.2! 2.8! 81! 2! CD25! 42! 7! 1! 7! 3! 67! WT! Vav-CreNfatc1αΑ fl/fl! 85! 9! B2! 14! 3! CD44! e! CD3! 5

6 Supplementary Figure 3 Defective T cell development in Vav-CreR-26-caNfatc1αA- Stop fl/fl (Vav-CreNfatc1αA fl/fl ) mice (a) Increase in CD4 - CD8 - CD44 - CD25 + DN3 and decreased CD4 - CD8 - CD44 - CD25 - DN4 cells in Vav-CreNfatc1αA fl/fl (n = 14) mice compared to WT (n = 6) or Vav-CreNfatc1αA fl/+ (n = 24) littermates. (b) Flow cytometry profiles of intracellular CD3ε levels in DN3 thymocytes from Vav-CreNfatc1αA fl/fl mice compared to WT mice. (c) Thymic cellularity in the Vav-CreNfatc1αΑ fl/fl Nfatc1 fl/fl mice compared to that in WT, Vav-CreNfatc1αA fl/fl and in Vav-CreNfatc1 fl/fl mice. Vav-CreNfatc1αΑ fl/fl Nfatc1 fl/fl mice only express NFATc1α from the knocked-in gene and lack NFATc1 expression from the endogenous gene. (d) Flow cytometry analysis of DN thymocytes from WT, Vav- CreNfatc1αA fl/fl, Vav-CreNfatc1 fl/fl and Vav-CreNfatc1αΑ fl/fl Nfatc1 fl/fl mice for the distribution of DN1 to DN4 cells. (e) Unimpaired B-lineage differentiation potential of Vav- CreNfatc1αA fl/fl DN1 cells. 4 x 1 4 sorted WT or Vav-CreNfatc1αA fl/fl DN1 cells were cocultured on monolayers of OP9 stromal cells in X-vivo medium supplemented with rhflt3 ligand (5 ng/ml) and rhil-7 (1 ng/ml) for 6 days. Subsequently, the cells were analyzed for differentiation into B (B2 + ) or T (CD3 + ) lineage by flow cytometry. Numbers within each plot represent percent respective population. Data are representative of three independent experiments and are shown as mean ± s.d., *** P =.6 and ** P =.36, Oneway ANOVA. 6

7 Supplementary Figure 4! a! Cells! WT DN4! 32! c! DN! DP! CD8 +! CD4 +! Isotype! Thymus! CD8 +! CD4 +! LN! CD5! Annexin V! b! Nfatc1!! Nfatc1"! Actb! CD48 (µg/ml)!.5 1 3! CD49d! % of Max FL2-H: -PE CD11a! CD18! CD62L! d! Mean fluorescence index (MFI)! 3 3 CD2! CD5! CD48! CD49d! CD11a! CD62L! P =.54! **! 15 5 P =.24! 1 **! 1 P =.1! ***! P =.2! **! P =.1! P <.1! ***! ***! 5 P <.1! ***! 1 3 P =.7! 25 ***! 15 5 P =.18! 15 **! 5 P <.1! P =.33! ***! **! 15 5 P =.1! ***! CD4 + CD25 -! CD4 + CD25 +! Thymus! LN! 7

8 Supplementary Figure 4 Differential expression of various integrins in thymocyte subsets and in T cells (a) Annexin V analysis to discriminate live and dead cells in WT DN4 cells cultured in complete RPMI-16 medium (1% FCS) for 12 h. (b) RT-PCR analysis for Nfatc1α and Nfatc1β expression in WT DN3 cells stimulated with indicated concentrations of CD48 Abs for 18 h. (c) Flow cytometry profiles of CD5, CD49d, CD11a, CD18 and CD62L expression on DN, DP, CD4 + and CD8 + SP T cells from thymus and in CD4 +, and CD8 + T cells from LNs of WT mice. (d) Expression of various integrins on CD4 + CD25 + regulatory T cells (T reg ) from thymus and LNs of WT mice (n = 4), compared to CD4 + CD25 - effector T (T eff ) cells. Histograms depict mean fluorescence intensity (MFI) for indicated integrin. Data are representative of three independent experiments and are shown as mean ± s.d., paired t- test. 8

9 Supplementary Figure 5! a! b! ! R3! CD4 + CD25 -! CD4 + CD25 int! CD4 + CD25 hi! 1 2 3! R2! CD25! 1 1 R1! 96! CD2! CD48! 49d! CD4! c! ! R3! CD11a! CD4 + CD25 -! CD4 + CD25 int! CD4 + CD25 hi! CD18! CD62L! CD25! CD4! 4! R2! R1 95!! WT! control! egfp! 9

10 Supplementary Figure 5 The level of integrin expression correlates with that of Nfatc1 expression (a) Dot plot showing the three distinct populations of thymic CD4 + T cells; CD4 + CD25 -, CD4 + CD25 lo and CD4 + CD25 hi from WT mice based on CD25 expression. (b) Flow cytometry profiles of indicated integrin expression levels on thymic CD4 + CD25 hi cells compared to CD4 + CD25 lo and CD4 + CD25 - cells from WT mice. (c) Flow cytometry profiles demonstrating the thymic CD4 + CD25 hi, CD4 + CD25 lo and CD4 + CD25 - cells, and their corresponding Nfatc1 expression levels as measured by GFP expression in Nfatc1-eGfp-Bac tg reporter mice. 1

11 Supplementary Figure 6! a! Color key! (Percent! methylation)!!++""" *+""" )+""" (+""" '+""" &+""" %+""" $+""" #+"""!+""" +""" Position! WT! of CpG! DN3! DN4!!""" "" #""" "" $""" "" %""" "" &""" "" '""" "" (""" "" )""" "" *""" ""!+""" ""!!""" ""!#""" ""!$""" ""!%""" ""!&""" ""!'""" ""!(""" ""!)""" ""!*""" "" #+""" "" #!""" "" ##""" "" #$""" "" #%""" "" #&""" "" #'""" "" #(""" "" #)""" "" #*""" "" b! Rag2 -/-" Rag2 -/-" anti-cd3" c! d! H3K4me1" H3K4me3" Pol II" H3K4me1" H3K4me3" Pol II" P1" NFAT" GATA" PU.1" Nfac1" P1" P2" E1" E2" Rel. Luc. Activity (x1 3 )" 1" 9" " 3" Luiferase pa E2" E3, E4 Activity Assay" P1"P2" E3" pa1 E1 " E2" pa2" E4" Ex " P1" " Luiferase pa E" Med" P+I" P1 P1-E2 P1-E3 P1-E4 " Rel. Luc. Activity (x1 3 )" 1" 9" " 3" "! Med" P+I" Wt N m123 G m Pu m! e! Chr18! RP23-361H16 (214kb)" Nfatc1-eGfp-Bac tg " " P1" P2" GFP" pa" pa1" E1" E2" pa2" " T7" Ex " - E2 (1Kb)" Nfatc1-eGfp-Bac-ΔE2 tg " SP6" P1" P2" GFP" pa" pa1" E1" pa2" T7" Ex " SP6" 11

12 Supplementary Figure 6 DNA methylation status at the Nfatc1 P 1 promoter in WT DN3 and DN4 cells (a) CpG methylation profiles of a 497 bp (-818 till -1297) segment of the murine Nfatc1 P1 promoter region from WT ptcr-negative DN3 and ptcr-positive DN4 cells. The extent of methylation on individual cytosine residues is as indicated by the color code. (b) ChIP-Seq analysis of Nfatc1 gene in acd3 Abs stimulated Rag2 -/- DN3 cells for epigenetic modifications at P1 and P2 promoter regions as well as at the putative regulatory elements compared to unstimulated cells. Blue rectangles represent the promoters, and the pink rectangles highlight the putative enhancer regions. (c) The positions of the putative regulatory elements E3 and E4 in the Nfatc1 locus, and their influence on Nfatc1 P1 promoter activity as demonstrated by luciferase reporter assays in EL-4 thymoma cells. (d) Analysis of putative transcription factor binding motifs in the E2 element and the effects of mutation in individual motifs on Nfatc1 P1 promoter inducibility as revealed by luciferase reporter assays in EL-4 cells. (e) Schematic diagram of the Nfatc1-eGfp-Bac- E2 transgene construct used to generate the reporter mice. A 1 kb region of the E2 element was deleted from the original Nfatc1-eGfp-Bac tg cassette to inactivate the enhancer function. 12

13 Supplementary Figure 7! Unstimulated! acd3 + acd28! Unstimulated! Unstimulated! acd3 + acd28! Unstimulated! Unstimulated! acd3 + acd28! Unstimulated! Unstimulated! acd3 + acd28! Unstimulated! CD4 +! B! CD4 +! B! CD4 +! B! CD4 +! B! 13

14 Supplementary Figure 7 Nfatc1 P 1 promoter activity is regulated by the E2 enhancer element. Whole cell extracts from 2 x 1 7 positively selected LN CD4 + T-cells either left unstimulated or stimulated with acd3 + acd28 Abs for 48 h, and freshly isolated CD19 + splenic B cells were resolved in 12% polyacrylamide gels to detect GFP (upper panel) and NFATc1α (lower panel) expression in lymphocytes. Immunoblot images were recorded in a Vilber Lourmat Fusion-SL imager and data were analyzed using the Fusion Capt. V16 software. The original peroxidase signals (right panel) and the converted negative image (right panel) of the same membranes with molecular weight markers are shown. Protein extracts were prepared from Nfatc1-eGfp-Bac tg (lanes 1-3) and from Nfatc1-eGfp-Bac- E2 tg (lanes 4-6) lymphocytes as indicated, i.e. unstimulated LN CD4 + T-cells (lanes 1, 4), 48h acd3 + acd28 stimulated LN CD4 + T-cells (lanes 2, 5) and unstimulated CD19 + B-cells (lanes 3, 6). 14

15 Supplementary Figure 8! a! WT! N3! Cam! N3 x Cam! 6.5! 87! 5! 82! 1! 1! 1! 7! CD4! 4.5! 2! 8! 5! 74! 6! 75! 8! CD8! b! WT! N3! Cam! N3 x Cam! 35! 2! 23! 1! 79! 8! 77! 7! CD25! 52! 11! 7! 6! 1! 3! 12! 4! CD44! c! Notch signaling DN3 cells Enhanced NFATc1 activity (NFATc1" + NFATc1!) Block in T cell development at the DN3 stage T cell! lymphopenia! CD4 + T cells CD8 + T cells TCR! rearrangement pt" expression ptcr signaling NFATc1" activity (P1 promoter) Lack of NFATc1" activity Failure to suppress Ptcra expression Constitutive ptcr signaling Differentiation block at CD44 - CD25 + stage Uncontrolled proliferation T-ALL development Limited proliferation, and differentiation DN4 cells Additional oncogenic events CD4 + T cells Positive and negative selection DP thymocytes (TCR" gene rearrangement) CD8 + T cells 15

16 Supplementary Figure 8 NFATc1 activity plays an essential role in promoting T cell differentiation and in preventing Notch-induced T-ALL development (a) Flow cytometry profiles reveal the distribution of thymocyte subsets based on CD4 and CD8 expression in WT, N3 tg, Cam and N3 x Cam double tg mice (b) DN1-DN4 cells distribution within DN thymocytes based on CD44 and CD25 expression in indicated mice. Numbers inside each plot represent percent respective population. Data in (a) and (b) are representative of three independent experiments (n = 3 per group per experiment). (c) Scheme showing the influence of ptcr signaling-induced NFATc1α activity in facilitating T cell differentiation and in preventing T-ALL development. DN3 cells upon receiving Notch signals express the ptcr on their surface. Subsequently, ptcr signaling induces NFATc1α activation, which promotes a limited proliferation followed by differentiation of ptcr-positive DN3 cells to the DN4 stage. Simultaneously, NFATc1α activity suppresses the expression of Ptcra once the DN3 cells have received optimal ptcr signals. DN4 cells further differentiate to DP thymocytes, which through the process of positive and negative selection give rise to a normal repertoire of CD4 + or CD8 + SP T cells. Alterations to this physiological process such as, enhanced NFATc1 activity due to NFATc1α expression in ptcr-negative DN3 cells will lead to severe T cell lymphopenia, or lack of NFATc1α activity in ptcr-positive DN3 cells in combination with other oncogenic events will result in the development of leukemia (T- ALL) due to uncontrolled proliferation. 16

17 Supplementary Table 1 LIST OF RT-PCR PRIMERS Gene Primer Sequence Product Size Actb Nfatc1α Nfatc1β Nfatc1A Nfatc1B Nfatc1C For: 5 -CCAGGTCATCACTATTGGCAAGGA-3 Rev: 5 -GAGCAGTAATCTCCTTCTGCATCC-3 For: 5 -ATGCCAAGCACCAGCTTTCCAGTCCCTT CC-3 For: 5 -ATGACGGGGCTGGAGCAGGACCCGGAG TTC-3 Rev: 5 -CCTCAGAGCTTAAGGTTAGAAAGACAG AGTTACC-3 Rev: 5 -ACGTATGATCTCATTTACTGCGGCTGTA G-3 Rev:5 -GGACAGCTACTGTTCAGATGTGGACTC AC-3 Nfatc1α: For: 5 -GGGAGCGGAGAAACTTTGC-3 (P1 activity) Rev: 5 -GATCTCGATTCTCGGACTCTCC-3 Nfatc1β: For: 5 -CGACTTCGATTTCCTCTTCGAG -3 (P2 activity) Rev: 5 - GATCTCGATTCTCGGACTCTCC-3 Nfatc1 For: 5 -GACTTCGATTTCCTCTTCGAGTTC-3 Rev: 5 -CTCGATTCTCGGACTCTCCAG-3 Nfatc2 For: 5 -GGGTTCGGTGAGTGACAGTT-3 Rev: 5 -CTCCTTGGCTGTTTGGGATA-3 Nfatc3 For: 5 -CCGATGACTACTGCAAACTGTGG-3 Rev: 5 -TTTGAATACTTGGGCACTCAAAGG-3 Notch1 For: 5 -TTGACGTCACTCTCCTGTGC-3 Rev: 5 -ACACAGGTGCCATTGTTGAA-3 Notch2 For: 5 - ACCCTTGTATGCACGGAGTC -3 Rev: 5 - CCAGGTTATTGCACGTTCCT -3 Notch3 For: 5 -GCACCTGCAACCCTGTTTAT-3 Rev: 5 -TCTCCAGCATCACCACAGAG-3 Ptcra For: 5 - TCACACTGCTGGTAGATGGA-3 Rev: 5 -TAGGCTCAGCCACAGTACCT-3 Rag1 For: 5 - ACCATGTGTCAAGCCACAAA-3 Rev: 5 -TGGCTACAGCTGAGGAAGGT-3 Rag2 For: 5 -TCTCTAAAGATTCCTGCTACCTC-3 Rev: 5 -TGGAATTCACTGCTGGGGTAC-3 Cd3e For: 5 - CCTGTTCCCAACCCAGACTA-3 Rev: 5 - AGGGCCAATTAGGAGAGGAA-3 Cd3z For: 5 - ATCCCAGGGAAGCAGAAGAT -3 Rev: 5 - TGTGCCGATCTCACTGTAGG -3 Gata3 For: 5 -TGAAGAAAGAAGGCATCCAG-3 Rev: 5 -AACTCTTCGCACACTTGGAG-3 Id1 For: 5 - CCAGTGGGTCTCATCCCTTA -3 Rev: 5 - AGAAATCCGAGAAGCACGAA -3 Id2 For: 5 - ACTCGCATCCCACTATCGTC -3 Rev: 5 - TCCCCATGGTGGGAATAGTA bp 25 bp 319 bp 311 bp 297 bp 371 bp 343 bp 8 bp 373 bp 37 bp 364 bp 332 bp 563 bp 412 bp 4 bp 3 bp 368 bp 453 bp 17

18 Runx1 For: 5 - GAGGCAAACTCTGTCCTGAA -3 Rev: 5 - TTAGGCCTCAAAGACACCTG -3 Bcl11b For: 5 - CCCCCAGCCTACAGATAAAT -3 Rev: 5 - CGGGTCAACAGAATTCAAAC -3 Ebf1 For: 5 - TGCGGAAATCCAACTTCTTC -3 Rev: 5 - GGTTCTTGTCTTGGCCTTCA -3 Pax5 For: 5 - GGGCTCCTCATACTCCATCA -3 Rev: 5 - CGTCAAGTTGGCTTTCATGT -3 Pou2f2 For: 5 - GGAGCTGGAACAGTTTGCTC -3 Rev: 5 - GATGCTGGTCCTCTTCTTGC -3 Sfp1 For: 5 - CGGATGACTTGGTTACTTACG -3 Rev: 5 - GTAGGAAACCTGGTGACTGAG -3 Csf1r For: 5 - ATGAGTCCCTCTTCACTCCG -3 Rev: 5 - ACCTTCAGCACTGCATCTTC -3 Cebpa For: 5 - CGCTGGTGATCAAACAAGAG -3 Rev: 5 - TCACTGGTCAACTCCAGCAC -3 E12 For: 5 - TGACAGCTACAGCAGGGATG -3 Rev: 5 - GAGTAGATCGAGGCCAGTGC -3 Cbfb For: 5 - GAAGCTGATGCTGACCTTGT -3 Rev: 5 - ACGCCAGCATTAAGACAGAC -3 Myb For: 5 - CTTCCAGCTTCAGCAAAGAG -3 Rev: 5 - GGAGGGTAAGGTAGGTGCAT -3 Ets1 For: 5 - TTCTCAGAAGCCTGTTGGAC -3 Rev: 5 - AAACAGTTTTGGACCCCTTC -3 Ets2 For: 5 - CTCAACACCGTCAATGTCAA -3 Rev: 5 - CTGGCTACAGTCCTCCTCAA -3 Adcy3 For: 5 - AGATGGAAACACGCTACTCG -3 Rev: 5 - AACATTGGCCATAACCAGAA -3 Pde3b For: 5 - CAGTAGCTTGATGGGTGCTT -3 Rev: 5 - AGACGATGACCTCTGCTTTG -3 Creb For: 5 - TGCCACATTAGCCCAGGTAT -3 Rev: 5 - GTACCCCATCCGTACCATTG -3 Foxp3 For: 5 - TCTCCAGGTTGCTCAAAGTC -3 Rev: 5 - CCAGGGGATAGTTCCTTGTT -3 Itga6 For: 5 - TGAGGTGTGTGAACATCAGG -3 Rev: 5 - TAGAGCCAGCATCAGAATCC -3 Itgav For: 5 - GGAGAACCAGAACCATTCCT -3 Rev: 5 - TTGCTCTTCTTGAGGTGGTC -3 Itgb1 For: 5 - AAGACATGGACGCTTACTGC -3 Rev: 5 - ATGGACCAGTGTCCAAAGAA -3 Itgb2 For: 5 - TAATGCAAGTTGCTGCATGT -3 Rev: 5 - GCTGGAGTCGTCAGACAGTT -3 Itgb3 For: 5 - ATACCAGGGAGGACCTTCAG -3 Rev: 5 - TCCTTCCCTGCTAGTTTCCT -3 Itgb4 For: 5 - GTCTGACGATCTGGACAACC -3 Rev: 5 - CGTTCTCCTTGCAGTTTGTT -3 Pecam1 For: 5 - TTGGCACAACAAACAAGCTA -3 Rev: 5 - GAAATCTTCTCGCTGTTGGA -3 Itga4 For: 5 - AAGCCAGCGTTCATATTCAG -3 Rev: 5 - ATCCAGCCTTCCACATAACA bp 311 bp 257 bp 321 bp 31 bp 292 bp 36 bp 499 bp 444 bp 32 bp 331 bp 29 bp 232 bp 39 bp 341 bp 56 bp 37 bp 36 bp 272 bp 267 bp 332 bp 257 bp 246 bp 292 bp 277 bp 18

19 Itga5 For: 5 - GTACCTGGGTGACAAGAACG -3 Rev: 5 - GTTCAGGTTCTTGCTGAGGA -3 Icam1 For: 5 - CCAAGAAACGCTGACTTCAT -3 Rev: 5 - CGACCCTTATGAGAAAAGCA -3 Icam2 For: 5 - GAAGCCACAGAGTCTTGGAA -3 Rev: 5 - TCAGTGTGACTTGAGCTGGA -3 Vcam1 For: 5 - CTGTACATCCCTCCACAAGG -3 Rev: 5 - ACACGTCAGAACAACCGAAT -3 Itgae For: 5 - AATGGCATTCAGTGGTCTGT -3 Rev: 5 - TCCTTGTGCTCTCCAAGTTC -3 Sell For: 5 - CGCTCATTCATCCCATTAAC -3 Rev: 5 - GCAAGGAGTCTGAGTTTCCA bp 327 bp 244 bp 321 bp 346 bp 224 bp 19