Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

<p>Abstract</p> <p>Background</p> <p>Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning.</p> <p>Results</p> <p>Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from <it>in vitro </it>fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping.</p> <p>Conclusion</p> <p>The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.</p

Somatic cell nuclear transfer in cattle

The development of somatic nuclear transfer procedures and the factors that are likely to affect the success of nuclear transplantation were discussed in the first chapter. In chapter 2, the effects of cell cycle stage and age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages and the in vitro life span of these cell lines were analyzed. The cell cycle characteristics of both fetal and adult cells were analyzed as a population of cells in culture. To study the individual cells from a population, a “shake off” method was developed to isolate G1 cells and evaluate progression through cell cycle. Irrespective of the age of the cell donor, the mean cell cycle length in isolated cells was shorter than what was observed for cells cultured as a population. In vitro development was analyzed after fusing confluent and isolated G1 cells to enucleated metaphase II oocytes. These results indicate that there were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells and confluent cells. Because previous studies do not clearly demonstrate the cell cycle stage of the donor cell that resulted in development to term, Chapter 2 evaluates the methods of producing populations of G0 and G1 cells and the cell cycle characteristics of G0 and G1 cells. Furthermore, in vitro and in vivo development of embryos derived from high confluent, G0 cells and “shake off” G1 cells were evaluated. These results indicate that the isolated G1 cells supported development to term better than confluent cells. Finally, in Chapter 3, pregnancy establishment, pregnancy loss and health of newborn calves were evaluated for nuclear transfer embryos derived from cells of different genotypes from dairy and beef breeds. In addition, the effect of overnight shipping of cloned embryos and the synchrony between the age of the embryo and the estrus cycle of the recipient female on development were assessed. The results indicate that genotype has an effect on development of nuclear transfer embryos

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

Background: Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the cumulative errors could reveal the epigenetic reprogramming blocks in animal cloning. Results: Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion: The present study provides adata set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying cumulative errors after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. © 2009 Rodriguez-Osorio et al; licensee BioMed Central Ltd

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

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

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

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

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