Moving toward a higher efficiency of microcell-mediated chromosome transfer

Abstract

Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs.

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

“…Other cell types and species, including human, can be employed as recipients in the cell fusion process, widening the variety of tissue types beyond fibroblasts (Hiratsuka et al 2015; Liskovykh et al 2016; Suzuki et al 2016). In addition, selectable markers such as neomycin resistance can be used instead of TK1, alleviating concerns about the perturbation of nucleotide synthesis pathways by HAT medium (Aoki et al 2014).…”

Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

“…Capable of introducing large DNA sequences into recipient cells, HACs have shown great potential in a wide range of applications, such as recombinant protein production, drug selection and gene therapy (108–111). However, the construction of HACs remains non-trivial: it requires cloning of either telomere sequences and/or alphoid DNA, the formation of HACs occurs at very low frequency and only in certain cell lines (112), and the transfer of HACs from donor cells into recipient cells is difficult (113, 114). Moreover, the presence of large telomere sequences and/or alphoid DNA on the HACs, and the heterchromatic state associated with these repeats, increases the likelihood of transgene silencing.…”

Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models

“…1a) because they are capable of micronucleation with elongated inhibition of microtubules [4] in response to colcemid [1] or TN16 and griseofulvin treatment [5]. Microcells are isolated by the centrifugation of cells forming micronuclei.…”