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1 Supplemental Figure 1 Supplemental figure 1. Generation of gene targeted mice expressing an anti-id BCR. (A,B) Generation of the VDJ aid H KI mouse. (A) Targeting Construct. Top: Targeting construct for aid rearranged VDJ H. Middle: Germline locus with indication of J1-4 J H gene segments and Cµ. Bottom: Integrated transgene in ES cells before Cre-mediated excision of LoxP flanked regions. Lines indicate terminal points of homologous recombination with germline locus. LoxP excision sites are indicated. tace, promoter for Cre Recombinase; Cre, Cre recombinase; PolIII, promoter for neomycin gene transcription; Neo, neomycin resistence gene; VDJ aid H, rearranged V(D)J H gene segments of anti-id Ab2-1.4 BALB/c B cell hybridoma. E, indicates EcoRV sites used for restriction digest and Southern blot analysis. Southern blot 3 probe is indicated by a grey box. (B) Southern blot of EcoRV digest of genomic BALB/c DNA from ES cell clone 1C11 probed with 3 probe. WT band is 14,8 kb and targeted band 9,1 Kb. (C,D) The VJ aid κ KI mouse. (C) Targeting construct. Top: targeting aid construct for rearranged VJ κ from Ab2-1.4 hybridoma. Middle: Germline locus with Jκ1-5 segments and Cκ indicated. Bottom: integrated transgene in ES cells. LoxP excision sites are
2 indicated. Lines indicate terminal points of homologous recombination with germline locus. PGK, promoter for neo. K, Kpn sites used for restriction digest and Southern blot. Southern blot 5 probe indicated by grey box. (D) Southern blot of Kpn digest of genomic BALB/c DNA from ES cell clone 1B12 probed with 5 probe. WT band is 45kb and targeted band is 8,9 kb.
3 Supplemental Figure 2 Supplemental figure 2. Nucleotide sequence of Ab2-1.4 κ chain cdna. A. IMGT V gene segment alignment with BALB/c genomic DNA (closest match, accession number KO2159). The predicted amino acid sequence is indicated above the nucleotide sequence, with the germline amino acid indicated below only for amino acid substitutions. A dash (-), indicates a position where Ab2-1.4 cdna is identical to genomic DNA. Nucleotide mutations are indicated in bold. CDR regions as annotated by IMGT are given below by aa sequence. B. IMGT J gene segment alignment (closest match, accession number V00777). The junction between V and J gene segments of Ab2-1.4 κ is composed of the germline V gene segment nucleotides CC together with germline J segment nucleotide g that result in a codon for proline, P.
4 Supplemental Figure 3 Supplemental figure 3. Nucleotide sequence of Ab2-1.4 H chain cdna. A. V segment alignment. IMGT alignment with C57BL/6 genomic DNA on chromosome 12 (accession number AC073939) is the closest possible match (genomic DNA for BALB/c is not accessible for comparison). Likely mutations are indicated by bold and black. The predicted aa sequence is indicated above the nucleotide
5 sequence, with the germline amino acid indicated below only for amino acid substitutions. A dash(-) indicates a position where Ab2-1.4 cdna is identical to genomic DNA. Nucleotide mutations are indicated with bold. CDR regions as annotated by IMGT are given by aa sequence. Non templated nucleotides in N1 and N2 regions are shown in bold and grey (see also B) B. D and J segment analysis for Ab2-1.4 H chain cdna. Alignment to closest BALB/c D and J segments are given with accession numbers. A dash(-), indicates a position where Ab2-1.4 cdna is identical to genomic DNA. In the junction between V and D (N1) and D and J segments (N2) of Ab2-1.4, non- templated nucleotides have been inserted; indicated in bold and grey (as in A. above). Supplemental Figure 4 Supplemental figure 4. Characterization of B cells in bone marrow (A) and in spleen (B). A. B cell subsets in bone marrow of BALB/c and anti-id DKI mice. B220-gated cells were classified as pre/pro (AA4.1 + IgM - ), immature (AA4.1 + IgM + ) or mature (AA4.1 - IgM + ) B cells. One representative mouse is shown. B and C. B cell subset characterization in spleen of one representative anti-id DKI mouse (B) and one BALB/c mouse (C). Anti Id + B220 + cells are shown in a AA4.1/CD23 plot. From this plot, the cell populations in each quadrant (UL, UR, LR, LL) were analyzed for CD21 and IgM expression. Subpopulations were defined as follows: T1 (AA4.1 + CD23 - CD21 - IgM + ), T2 (AA4.1 +, CD23 + CD21 lo, IgM hi ), T3 (AA4.1 +, CD23 +, CD21 lo IgM int ), follicular, FO ( AA4.1 - CD23 + CD21 lo IgM + ), marginal zone, MZ (AA4.1 -, CD23 -, CD21 hi IgM hi ) and B1 (AA4.1 -, CD23 -, CD21 int, IgM int ). B: B cell subset characterization for B220 + cells in BALB/c. The data is summarized in Fig.1F.
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2. In Figure 10-4, why is edna made only from mrna and not also from trnas and ribosomal RNAs? Answer: edna is made from mrna and not from trnas or rrnas because polyt primers are used to prime the first
Supplemental Figure legends Figure S1. (A) (B) (C) (D) Figure S2. Figure S3. (A-E) Figure S4. Figure S5. (A, C, E, G, I) (B, D, F, H, Figure S6.
Supplemental Figure legends Figure S1. Map-based cloning and complementation testing for ZOP1. (A) ZOP1 was mapped to a ~273-kb interval on Chromosome 1. In the interval, a single-nucleotide G to A substitution
Historical Perspective
Genetic transformation of E.Coli and selection, DNA recombination without ligase: topoisomerase, cre-lox recombination, Gate way method etc. DNA library: genomic library, cdna library, expression library,
American Society of Cytopathology Core Curriculum in Molecular Biology
American Society of Cytopathology Core Curriculum in Molecular Biology American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 3 Molecular Techniques Separation and Detection, Part