Adding X and Y chromosomes to human cells
In this Web site, we have discussed how to make female sperm and male eggs. Both processes require the equivalent of adding all or part of a Y chromosome to an initially "female"-XX cell, or adding all of an X chromosome to an initially "male"-XY cell. The question thus arises: "How do we add human chromosomes to human cells"? Interesting, the question has two answers: microcell-mediated chromosome transfer (MMCT), and human artificial chromosomes (HACs). In this Web page, we first the MMCT process, and then HACs, providing lots of links to medical journal articles.
What is a chromosome? - Most of a chromosome is DNA - your genes plus filler material. At the end of the chromosome are protective caps known as telomeres (kind of like those plastic caps at the ends of your shoelaces). Chromosomes have central hubs, centromeres, to anchor the DNA. And chromosome have Origins-of-Replication (ORIs), a sequence of genetic instructions that allows the chromosome to copy/replicate itself.
Microcell-Mediated Chromosome Transfer
In 1977, R. Fournier and F. Ruddle at the Biology Department of Yale University, published a technique for transferring entire chromosomes between the cells of mammals. The process they invented is known as "microcell-mediated chromosome transfer" (MMCT), because it involves taking regular cells with an entire complement of chromosomes (such as the 46 chromosomes of humans) and chemically breaking up the full cell into microcells, that is, cells that contain 1 to a few chromosomes, preferably one chromosome. Then with cell sorting techniques, many of the microcells with one specific chromosome, such as the X or Y, are isolated into a separate sample. These microcells are then fused with regular cells, so that the resulting cells have an extra chromosome.
Fournier and Ruddle, in their published paper, documented how they transferred chromosomes from a mouse to the cells of other mice, Chinese hamsters and human cells. Their article is titled "Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells", Proc. National Academy of Sciences, January 1977, 319-323. ( (Abstract and citations). ( Abstract and full PDF). Three years later, C. McNeill and R. Brown of the Department of Cell Biology at the University of Texas in Houston published a paper transferring a chromosome in the other direction, from a human to a mouse cell. Their article is titled "Genetic manipulation by means of microcell-mediated transfer of normal human chromosomes into recipient mouse cells", Proc. National Academy of Sciences, September 1980, 5394-5398. ( (Abstract and citations). ( Abstract and full PDF). Of course, transferring chromosomes between cells of the same species (e.g., human to human) is even easier.
MMCT is usually a five-step process as follows. First, cells from which the X or Y chromosomes are to be obtained are treated with the drugs colcemid and colchicines, which arrests the cells in the metaphase stage, with prolonged exposure intefering with mitotic spindle formation, and causing cells to multinucleate and form nuclear membranes around individual or small numbers of chromosomes. Second, the treated cells are centrifuged in the presence of cytochalasin B, which disrupts microfilaments and prevents cells from returning in interphase, leading to microcell extrusion. Third, the microcells are collected and filtered through membranes with pore sizes as small as 3 micrometers, to select for microcells containing a single chromosome. Fourth, these microcells are fused to recipient cells, typically by adding phytohemagglutinin-P (PHA-P) to cause cell agglutination and polyethylene glycol (PEG) to dissolve cell membranes. Fifth, fused cells are selected for those cells containing the desired single chromosome, such as the X or Y, typically using chromosome probes to facilitate the selection.
In the 30 years since this foundational work, hundreds of papers have been published on the MMCT process, including papers on transferring human X and Y chromosomes, with the transfer techniques becoming ever more efficient. A bibliography of some of these papers and patents appears below. To help fertility clinics offer services to prepare female sperm and male eggs, most likely specialty companies will be established to cost effectively produce supplies of human microcells with single X and Y chromosomes.
Both as social comment and artistic creation, in 2000, the artist Eduardo Kac created the GFP Bunny by inserting a gene for Green Flourescent Protein into the embryo of a rabbit. When will the latest fashion trend be to have flourescent humans?
Human Artificial Chromosomes
Many years ago, scientists proposed a variety of approaches to making artificial chromosomes: remove DNA from existing chromosomes, induce cells to generation empty chromosomes, or chop up and reassemble chromosomes minus the DNA. You would have generic empty chromomsomes. If you could selectively add genes (such as sperm making genes), you would have a human artificial chromosome - a HAC (more commonly referred to as mammalian artificial chromosome, MAC, but let's face it, it is the human application that is of most fun and profit). This idea was not that strange, since yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) were already being used. But these types of artificial chromosomes have parts from non-humans (though as compared to humans with pig valves in their hearts), and can carry only small sized genes, a problem for some of the large human genes one might want to place inside a HAC.
A group of Hungarian scientists have induced cells to generate human artificial chromosomes. They generate human satellite DNA-based artificial chromosomes (SATACs - hilarious acrohym) by amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Such chromosomes can be made and purified in quantity, can carry large sequences of DNA, and can be transferred into cells and embryos of differenet species. The work of the Hungarian scientists is now being commercialized by a Canadian company, Chromos, which is now offering human artificial chromosome products. Click here for an overview of their technology, or a local copy.
Since the, other approaches to human artificial chromosomes have been developed, one or more of which will be useful for adding parts of the Y chromosome to female germ cells. A bibliography of papers and patents on artificial chromosomes appears below.
Bibliography for Microcell-Mediated Artificial Chromosomes
"The manipulation of chromosomes by mankind: the uses of microcell-mediated chromosome transfer", Chromosoma, 2005 ( Abstract )
"The human Y chromosome [transferred by MMCT] suppresses the tumorigenicity of PC-3, a human prostate cancer cell line, in athymic mice", Genes Chromosomes Cancer, 2005 ( Abstract )
"Microcell-mediated chromosome transfer (MMCT): small cells with huge potential", Mammalian Genome, 2003 ( Abstract )
"Multiple human chromosomes carrying tumor-suppressor functions for the mouse melanoma cell line B16-F10, identified by microcell-mediated chromosome transfer", Mol. Carcinogenesis, 2002 ( Abstract )
"Suppression of metastasis of rat prostate cancer by introduction of human chromosome 13", Asian J. Andrology, 2002 ( Abstract [uses MMCT])
"Microcell-mediated transfer of chromosome 4 into HeLa cells suppresses telomerase activity", Genes Chromosome Cancer, 2001 ( Abstract )
"Stability of transferred human chromosome fragments in cultured cells and in mice", Chromosome Research, 2000 ( Abstract )
"Telomerase-independent senescence of human immortal cells induced by microcell-mediated chromosome transfer", Mol. Carcinogenesis, 1999 ( Abstract )
"Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome", Mammalian Genome, 1999 ( Abstract )
"Simultaneous transfer of mitochondrial DNA and single chromosomes in somatic cells: a novel approach for the study of defects in nuclear-mitochondrial communication", Human Molecular Genetics, 1998 ( Abstract )
"Functional expression and germline transmission of a human chromosome fragment in chimaeric mice", Nature Genetics, 1997 ( Abstract )
"Evaluation of phenotypic alteration by microcell-mediated chromosome transfer", Anal Biochem., 1996 ( Abstract )
"Regulation of tissue-specific gene expression in microcell hybrids", Methods, 1996 ( Abstract )
"Stable oncogenic transformation induced by microcell-mediated gene transfer", Sci. China B., 1995 ( Abstract )
"Irradiation microcell-mediated chromosome transfer (XMMCT): the generation of specific chromosomal arm deletions", Genes Chromosomes Cancer, 1990 ( Abstract )
"General protocol for microcell-mediated chromosome transfer", Somat Cell Mol Genet., 1987 ( Abstract )
"Transfer and selective retention of single specific human chromosomes via microcell-mediated chromosome transfer", Methods Enzymol., 1987 ( Abstract )
"Selective transfer of individual human chromosomes to recipient cells", Mol Cell Biology, 1985 ( Abstract )
"A general high-efficiency procedure for production of microcell hybrids", Proc. Natl. Acad. Sciences, 1981 ( Abstract )
"Genetic manipulation by means of microcell-mediated transfer of normal human chromosomes into recipient mouse cells", Proc. Natl. Acad. Sciences, 1980 ( Abstract )
"Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells", Proc. Natl. Acad. Sciences, 1977 ( Abstract )
Bibliography for Human Artificial Chromosomes
"Human artificial chromosomes: potential applications and clinical considerations", Pediatr Clin North America, 2006 ( Abstract )
"An artificially constructed de novo human chromosome behaves almost identically to its natural counterpart durig metaphase and anaphase in living cells", Mol Cell Biol, 2006 ( Abstract )
"Human artificial chromosomes constructed using the bottom-up strategy are stably maintained in mitosis and efficiently transmissible to progeny mice", J. Biol. Chem., 2006 ( Abstract ) [uses MMCT]
"Progress in artificial chromosome technology", Biochem Soc Trans, 2006 ( Abstract )
"Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome", J. Human Genetics, 2006 ( Abstract ) [uses MMCT]
"A novel expression system for genomic DNA loci using a human artificial chromosome vector with transformation-associated recombination cloning", J. Human Genetics, 2005 ( Abstract )
"A novel human artificial chromosome vector provides effective cell lineage-specific transgene expression in human mesenchymal stem cells", Stem Cells, 2005 ( Abstract )
"Artificial and engineered chromosomes: developments and prospects for gene therapy", Chromosoma, 2005 ( Abstract )
"Artificial and engineered chromosomes: non-integrating vectors for gene therapy", Trends Mol Medicine, 2005 ( Abstract )
"Construction of a novel expression system on a human artificial chromosome", Biochem Biophys Res Communications, 2005 ( Abstract )
"Human artificial chromosome (HAC) vector provides a long-term therapeutic transgene expression in normal human primary fibroblasts", Gene Therapy, 2005 ( Abstract ) [uses MMCT]
"Construction of a novel human artificial chromosome vector for gene delivery", Biochem Biophys Res Communications, 2004 ( Abstract )
"Transfer and stable transgene expression of a mammalian artificial chromosome into bone marrow-derived human mesenchymal stem cells", Stem Cells, 2004 ( Abstract )
"Artificial chromosome vectors and expression of complex proteins in transgenic animals", Theriogenology, 2003 ( Abstract )
"Advances in human artificial chromosome technology", Trends Genetics, 2002 ( Abstract )
"Chromosome engineering: prospects for gene therapy", Gene Therapy, 2002 ( Abstract )
"Cloned transchromosomic calves producing human immunoglobin", Nature Biotechnology, 2002 ( Abstract ) [uses HACs via MMCT]
"Strategies for engineering human chromosomes with therapeutic potential", J. Gene Med., 2002 ( Abstract )
"Functional complementation of a genetic deficiency with human artificial chromosomes", Am J Human Genetics, 2001 ( Abstract )
"Stability of transferred human chromosome fragments in cultured cells and in mice", Chromosome Research, 2000 ( Abstract )
"Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts", Nature Biotechnology, 2000 ( Abstract )
"Making CENs of mammalian artificial chromosomes", Mol Genet Metab, 1999, ( Abstract )
"Mammalian artificial chromosomes as tools for gene therapy", Current Opinions in Genetic Development, 1998 ( Abstract )
"Formation of de novo centromeres and construction of first-generation human artificial minichromosomes", Nature Genetics, 1997 ( Abstract )