Assessment of genome integrity with array CGH in cattle transgenic cell lines produced by homologous recombination and somatic cell cloning

<p>Abstract</p> <p>Background</p> <p>Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that multiple rounds of cloning compromise the genome integrity or/and introduce epigenetic errors in the resulting cell lines, rendering a decline in cloning. To test these possibilities, we performed 9 high density array Comparative Genomic Hybridization (CGH) experiments to test the genome integrity in 3 independent bovine transgenic cell lineages generated from genetic modification and cloning. Our plan included the control hybridizations (self to self) of the 3 founder cell lines and 6 comparative hybridizations between these founders and their derived cell lines with either high or low cloning efficiencies.</p> <p>Results</p> <p>We detected similar amounts of differences between the control hybridizations (8, 13 and 39 differences) and the comparative analyses of both "high" and "low" cell lines (ranging from 7 to 57 with a mean of ~20). Almost 75% of the large differences (>10 kb) and about 45% of all differences shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but caused by systematic factors associated with local genomic features (e.g. GC contents).</p> <p>Conclusions</p> <p>Our findings reveal that large copy number variations are less likely to arise during genetic targeting and serial rounds of cloning, fortifying the notion that epigenetic errors introduced from serial cloning may be responsible for the cloning efficiency decline.</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

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

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

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