FISH analysis of DT40 clones carrying the corrected modified human chromosomes.

<p>For the hybrid DT40 clone SLK2, the presence of both hChr2 and hChr22 was detected by human COT-1 DNA as the probe; for DT40 clones carrying the translocated human chromosomes, a two-color FISH assays were conducted: the hChr2 painting probe was labeled with Rhodamine and the hChr22 painting probe was labeled with Fluorescein.</p

Modification of hChr14.

<p>(A) A lox511 sequence along with the promoter-less <i>puro</i> cassette was integrated at the AL512355 locus with gene targeting vector p14CEN(FR)hygpuro^lox511DT, and a lox511 sequence along with a CAG promoter and a hygromycin (hyg) selection cassette was integrated at locus AL512355 with gene targeting vector pSC355CAG^lox511hisDDT. Following Cre expression, the ~85 Mb genomic sequence was removed rendering <i>puro</i> expression. A loxP sequence and a GFP reporter cassette was then integrated at the <i>RNR2</i> locus to generate 14D. (B) FISH analysis of a DT40 clone, 14D, containing the correctly modified hChr14.</p

Bovinization of hIgM CH2-TM Domain.

<p><b>The upper line shows the genomic configuration of the CH 1-TM2 domain of hIgM, the middle line shows the gene targeting vector pCH2CAGzeoDT, and the bottom line depicts the modified CH2-TM2 domain of hIgM in a DT40 cell clone, CH2D4</b>. </p

Human IgG production profile in cKSL-HACΔ/DKO Tc cattle. (A) Comparison of average total hIgG (left panel) and fully hIgG (hIgG/hIgκ) concentrations in the sera of κHAC/DKO and cKSL-HACΔ/DKO calves at 5-6 months of age. (B) IgG subclass distribution in the plasma of cKSL-HACΔ/DKO calves. (C) Mean fluorescence intensity (MFI) of tumor cells stained with the sera from immunized cattle. Left panel shows the MFI with anti-hIgG antibodies (measuring total hIgG); right panel shows the MFI with anti-hIgκ antibodies (measuring fully hIgG/hIgκ).

<p>Human IgG production profile in cKSL-HACΔ/DKO Tc cattle. (A) Comparison of average total hIgG (left panel) and fully hIgG (hIgG/hIgκ) concentrations in the sera of κHAC/DKO and cKSL-HACΔ/DKO calves at 5-6 months of age. (B) IgG subclass distribution in the plasma of cKSL-HACΔ/DKO calves. (C) Mean fluorescence intensity (MFI) of tumor cells stained with the sera from immunized cattle. Left panel shows the MFI with anti-hIgG antibodies (measuring total hIgG); right panel shows the MFI with anti-hIgκ antibodies (measuring fully hIgG/hIgκ).</p

Flow cytometry analysis of B cell development in cKSL-HACΔ/DKO Tc cattle. The percentages of IgM-single positive (IgM⁺) and IgM/CD21-double positive (IgM⁺/CD21⁺) B cells in PBMCs from newborn animals were compared between κHAC/DKO and cKSL-HACΔ/DKO calves.

<p>Flow cytometry analysis of B cell development in cKSL-HACΔ/DKO Tc cattle. The percentages of IgM-single positive (IgM⁺) and IgM/CD21-double positive (IgM⁺/CD21⁺) B cells in PBMCs from newborn animals were compared between κHAC/DKO and cKSL-HACΔ/DKO calves.</p

Construction of cKSL-HACΔ.

<p>DT40 clones carrying modified hChr14 (CH2D4) and modified hChr2/hChr22 (SLKD18), respectively, were fused by WCF to generate DT40 hybrid clone cKSLD22. The presence of the two modified human chromosomes in cKSLDH22 clone was confirmed by FISH with human COT-1 DNA as the probe. Transfection of cKSLDH22 clone with Cre-expression plasmid induced for site-specific translocation between the two loxP sites at the <i>cos138</i> locus on the SLK HAC and another at the <i>RNR2</i> locus on the CH2D HAC. The presence of the three human immunoglobulin loci, h<i>IgH</i>, h<i>IgK</i> and h<i>IgL</i>, as a single HAC was confirmed by three-color FISH, with the DNA probes for h<i>IgH</i>, h<i>IgK</i> and h<i>IgL</i> labeled with SpectrumRed, SpectrumGreen, and SpectrumOrange, respectively.</p

Translocation between hChr22 and hChr2. A DT40 clone (K53) carrying the previously modified hChr2 fragment is fused with a DT40 clone (STL54) carrying the previously modified hChr22 fragment via WCF to generate DT40 hybrid clone SLK2. Transfection of SLK2 by Cre-expression plasmid induces the recombination between the two lox2272 sites, one at the AC104134 locus on the hChr2 and another at AP000553 locus on the hChr22. The translocated chromosome (carried by DT40 clone SLKH6) was transferred to plain DT40 cells by MMCT to generate clone SLKD18. The lower panel of the figure depicts the Cre/loxP mediated DNA recombination event. Note that only the correctly DNA recombination event activates the <i>puro</i> gene.

<p>Translocation between hChr22 and hChr2. A DT40 clone (K53) carrying the previously modified hChr2 fragment is fused with a DT40 clone (STL54) carrying the previously modified hChr22 fragment via WCF to generate DT40 hybrid clone SLK2. Transfection of SLK2 by Cre-expression plasmid induces the recombination between the two lox2272 sites, one at the AC104134 locus on the hChr2 and another at AP000553 locus on the hChr22. The translocated chromosome (carried by DT40 clone SLKH6) was transferred to plain DT40 cells by MMCT to generate clone SLKD18. The lower panel of the figure depicts the Cre/loxP mediated DNA recombination event. Note that only the correctly DNA recombination event activates the <i>puro</i> gene.</p

Diagrams of pre-BCRs in κHAC/DKO and cKSL-HACΔ/DKO cattle.

<p>Protein molecules/domains encoded by human genes are drawn in red while bovine in blue. The dotted double-lines represent B cell membrane.</p

Modification of hChr22. The intact hChr22 carried in a DT40 cell line, 52-18, was first truncated at the AP000350 locus with the targeting vector pTELCAGzeoSLFR. A DT40 clone, ST13, carried the truncated hChr22 was transfected with targeting vector p553CAG^lox2272bsrDT to integrate the lox2272 and the CAG promoter at the locus AP000553. The distance between AP000350 and AP000553, where the h<i>IgL</i> and h<i>SLC are</i> located, is about 2 Mb.

<p>Modification of hChr22. The intact hChr22 carried in a DT40 cell line, 52-18, was first truncated at the AP000350 locus with the targeting vector pTELCAGzeoSLFR. A DT40 clone, ST13, carried the truncated hChr22 was transfected with targeting vector p553CAG^lox2272bsrDT to integrate the lox2272 and the CAG promoter at the locus AP000553. The distance between AP000350 and AP000553, where the h<i>IgL</i> and h<i>SLC are</i> located, is about 2 Mb.</p

Modification of hChr2.

<p><b>A </b><b>previously </b><b>engineered </b><b>hChr2 </b><b>fragment </b><b>containing </b><b>the </b><b>entire </b><b>h<i>IGK</i></b><b>locus </b><b>in </b><b>a </b><b>DT40 </b><b>clone, κTL1</b> [12]<b>, was </b><b>further </b><b>modified </b><b>with </b><b>gene </b><b>targeting </b><b>vector </b><b>pTEL’hisDpuro</b>^{<b>lox2272</b>}<b>F9R9 </b><b>to </b><b>both </b><b>truncate </b><b>the </b><b>hChr2 </b><b>fragment</b> and <b>integrate </b><b>the </b><b>lox2272</b> and <b>the </b><b>promoter-less </b><b><i>puro</i></b><b>gene </b><b>at </b><b>the</b> AC104134 <b>locus</b>. <b>The DT40 clone carrying the truncated form of hChr2 was named as K53. The upper panel of the figure shows the structures of hChr2 before and after truncation. The lower panel of the figure depicts the homologous recombination event mediated by the pTEL’hisDpuro^lox2272F9R9 gene targeting vector.</b></p