Analysis of constructed isKcHACΔ vector.

<p>(<i>A</i>) Extensive genomic PCR for genotyping of the isKcHACΔ vector. Location of each genomic PCR primer pair is depicted in relation to the isKcHACΔ vector structure. (<i>B</i>) CGH analysis among three different CHO clones containing the isKcHACΔ vector. DNA from isKCDC15-8 was used as a reference. There was no apparent structural difference in the isKcHACΔ among the three cell lines.</p

Trans-class switched bovine IgG levels in a series of Tc cattle.

<p>(A) The ratio of t-bIgG to total IgG (human IgG and t-bIgG) in percentage. The number of Tc <b>cattle</b> evaluated at each time points was noted on top of each bar in brackets. (B) Serum t-bIgG concentration in Tc bovines. The serum t-bIgG concentrations of Tc bovines with TKO background were evaluated at ages of 5–6 months and at 11–12 months. The number of Tc bovines evaluated at each time points was noted on top of the each bar in brackets.</p

The chromosome engineering strategy including pedigree and deduced structure of bovine chromosomes for knockouts is shown.

<p>(A) Breeding pedigree to establish G0 TKO cell lines. Male cell line 6939 and female cell line 3427 were sequentially targeted to obtain <i>IGHM ⁻</i>^/+<i>IGHML1⁻</i>^/<i>−</i> and <i>IGHM⁻</i>^/<i>−</i> animals, respectively, for the first round of breeding, which generated both male and female G0 <i>IGHM⁻</i>^/<i>−</i><i>IGHML1⁻</i>^/+ cell lines. These cell lines were subjected to the cluster deletion to generate <i>IGHM^−/−IGHML1^−/+IGL^−/+</i> animals for the second round of breeding, which led to establishment of male and female G0 TKO cell lines. (B) Deduced structure of the bovine <i>IGH</i> gene cluster on bChr21. BAC clone 227-A16 contained part of the <i>IGH</i> variable region and the <i>IGHML1</i> through the C_γ1 region. BAC includes the C_γ2 through the 3′E_α region. The size of the three BAC clones contiguously is estimated to be around 380 kb. (C) Genomic organization of the bovine <i>IGLJ-IGLC</i> gene cluster on bChr17.</p

Construction of the istHAC vector.

<p>A flow of the istHAC vector construction is illustrated. The <i>attP</i> sequence is integrated at 5’ side of the hI_γ1 exon 1 and 3’ side of the h<i>IGHG1</i> TM2 by the targeting vectors phI_γ1FRTCAGattPhisDDT and ph_γ1TMNeoattPDT, respectively. Then, the replacement vector pBAC-istHAC is introduced with the ΦC31 recombinase to bring about the <i>attP</i>/<i>attB</i> recombination to replace the flanked region. The successful replacement causes the CAG promoter-driven DsRed gene to be reconstituted to provide red fluorescence for sorting. Finally, the DsRed cassette is removed by the FLP expression.</p

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle

<div><p>Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.</p></div

Construction of the isHAC and isKcHACΔ vectors.

<p>A flow of the isHAC and isKcHACΔ vector construction are illustrated. The bovinizing vector pCC1BAC-isHAC is a BAC-based one (backbone is pCC1BAC vector), consisting of 10.5 kb and 2 kb of genomic DNA as a long and short arm, respectively, 9.7 kb of the bovine genomic DNA covering the bovine I_γ1-S_γ1 and its surrounding region to replace the human corresponding 6.8 kb of I_γ1-S_γ1 region, the chicken β-actin promoter-driven <i>neo</i> gene flanked by <i>FRT</i> sequence and the <i>DT-A</i> gene. After the targeted bovinization, the <i>neo</i> cassette is removed by FLP introduction.</p

Representative flow cytometry analysis of PBMCs from a series of cKSL-HACΔ/DKO and cKSL-HACΔ/TKO calves at birth is shown.

<p>For IgM detection, anti-hIgM antibody was used. From left to right panels, PBMCs were stained with IgM alone, IgM/bCD21, IgM/bλ, IgM/bκ and IgM/hκ antibodies. Each red number represents % of cells in Q1 (IgM alone) or Q2 (IgM/bCD21, IgM/bλ, IgM/bκ and IgM/hκ).</p

Characterization of κHAC/DKO, cKSL-HACΔ/DKO and KcHAC/DKO cattle by PBMC expression profile and serum IgG profile.

<p>(<i>A</i>) Representative flow cytometry analysis of PBMCs from a series of HAC/DKO calves at newborn stage. For IgM detection, anti-hIgM or anti-bIgM antibody was used. From left to right panels, PBMCs were stained for IgM alone, IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ. Each red number represents percentages of cells in Q1 (IgM alone) or Q2 (IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ). NA: not applicable (because, at that time, the anti-bIgκ antibody was not available). (<i>B</i>) Box-whisker plots of serum concentrations of total hIgG (g/L)(top left), fully hIgG/hIgκ (g/L)(top right), serum fully hIgG/hIgκ (%)/total hIgG (bottom right) and hIgG1/hIgG2 ratio (bottom left) in a series of HAC/DKO cattle at 5–6 months of age. F, cKSL-HACΔ/DKO (n = 33); G, KcHAC/DKO (n = 12); H, κHAC/DKO (n = 8). Dots represent outliers. The value of calf 468 were indicated with arrows.</p

<i>IGHML^−/−IGHM^−/+</i> (male) and <i>IGHM^−/−</i> (female) calving efficiencies.

<p><i>IGHML^−/−IGHM^−/+</i> (male) and <i>IGHM^−/−</i> (female) calving efficiencies.</p

Characterization of isHAC/TKO, istHAC/TKO and isKcHACΔ/TKO cattle by PBMC expression profile.

<p>Representative flow cytometry analysis of PBMCs from a series of HAC/TKO calves at newborn stage. For IgM detection, anti-hIgM or anti-bIgM antibody was used. From left to right panels, PBMCs were stained for IgM alone, IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ. Each red number represents percentages of cells in Q1 (IgM alone) or Q2 (IgM/bCD21, IgM/bIgλ, IgM/bIgκ and IgM/hIgκ).</p