Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Synaptonemal complex analysis of domestic sheep (Ovis aries) with Robertsonian translocations. II. Trivalent and pairing abnormalities in Massey I and Massey II heterozygotes

Zygotene and pachytene spermatocytes from Massey I (t1 5;26) and Massey II (t2 8;11) translocation heterozygotes each contained one trivalent, often delayed in pairing, while cells from double Massey translocation heterozygotes had two such trivalents. As meiosis progressed, trivalents became fully paired, with acrocentric axes in a cis configuration. Abnormal pairing configurations often resulted from interactions between unpaired chromosome axes or segments. However, when two Massey trivalents were present in the same nucleus, there was no pairing interaction between them. In different Massey translocation heterozygotes, trivalent-involved pairing abnormalities occurred in 14–28% of cells, with XY–trivalent and XY–bivalent–trivalent associations being as high as 7.1–23.1%. In spermatocytes from single and double Massey translocation heterozygotes with normal-sized testes, the total SC abnormality frequency was 34.4% for the t1 heterozygotes, 27.1% for the t2 heterozygotes, and 21.4% for the double heterozygote. One Massey II heterozygote with one normal and one small testis had significantly higher SC abnormality frequency (54%) than normal rams. A trisomic cell was recorded in one ram and two hyperdiploid cells in another ram, but these were unrelated to the translocations. It is suggested that resolution of pairing abnormalities by synaptic adjustment is important in reducing the effects on fertility of the translocations.Key words: sheep, Robertsonian translocation, trivalent, abnormal pairing configuration.

Analysis of protein patterns in two-dimensional gels of cultured human cells with trisomy 21.

The occurrence of one chromosome of the cell in triplicate (trisomy, Ts) should increase the amount of all cell proteins coded by genes located on this chromosome. Many other proteins should be altered in their quantity by regulator genes, present in a threefold dosage, and by several indirect effects of the trisomy. We used two-dimensional electrophoresis to investigate the effect of the human Ts 21 on the proteins. Cells from different individuals with Ts 21 were cultured. Seven cell lines were derived from skin tissue and four from cells in amniotic fluid, and two cell clones were raised from fetal lung with Ts 21 mosaic. Stained two-dimensional protein patterns from these cell lines were compared with control patterns, and clearly visible differences in the staining intensity of corresponding polypeptide spots were evaluated. A few quantitatively variant polypeptides occurred in all trisomic cell lines investigated that were of a particular cell type, but no variants were consistently present in all of the 13 cell lines investigated. However the total number of variants (including variants not found in all individual cell lines) was considerably higher in trisomic cells than in normal cells.