Marilyn Monk

Human embryonic genes re-expressed in cancer cells.

Human preimplantation embryonic cells are similar in phenotype to cancer cells. Both types of cell undergo deprogramming to a proliferative stem cell state and become potentially immortal and invasive. To investigate the hypothesis that embryonic genes are re-expressed in cancer cells, we prepare amplified cDNA from human individual preimplantation embryos and isolate embryo-specific sequences. We show that three novel embryonic genes, and also the known gene, OCT4, are expressed in human tumours but not expressed in normal somatic tissues. Genes specific to this unique phase of the human life cycle and not expressed in somatic cells may have greater potential for targeting in cancer treatment.

Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos.

Mouse preimplantation embryos were accurately diagnosed as normal or mutant at the beta-major haemoglobin locus by amplification of specific DNA sequences in a single cell. A DNA sequence containing the whole of exon 3 and some 3 untranslated sequences within the beta-major haemoglobin gene was amplified in single blastomeres by means of the polymerase chain reaction (PCR). Blastomeres were removed from embryos of four to eight cells from normal BALB/c mice and from mutant (thalassaemic) BALB/c mice homozygous for a deletion of the whole beta-major haemoglobin gene. The sensitivity of the amplification procedure was enhanced by the sequential use of two sets of oligonucleotide primers for 30 cycles of amplification each, the second pair being located within the segment amplified by the first pair. The product (204 base-pairs) could be easily visualised in ethidium bromide-stained agarose gels. Stringent precautions to prevent contamination were taken, and with these precautions the PCR amplification procedure could be carried out under normal laboratory conditions. These procedures for diagnosis of genetic disease before implantation should be applicable to preimplantation diagnosis of any monogenic disorder in man for which the affected DNA sequence is known.

Amplification of a β-haemoglobin sequence in individual human oocytes and polar bodies

A 680 base-pair sequence of the human beta-haemoglobin gene was reproducibly amplified in individual unfertilised human oocytes and in first polar bodies isolated from them. Specificity and sensitivity of amplification were achieved by two sequential reactions with two sets of primers, amplifying first a 725 base-pair sequence and secondly a 680 base-pair sequence from within the first amplified fragment. A restriction enzyme digest of the DNA amplified from a single oocyte with the endonuclease Dde I confirmed the identity of the amplified beta-haemoglobin fragment; this technique provides a diagnostic test for the genetic defect responsible for sickle cell anaemia. Analysis of the DNA from the first polar body may enable detection of such defects in unfertilised eggs from carrier women. Selection of eggs without the defect for fertilisation may therefore obviate the need for diagnostic procedures on embryos.

PREIMPLANTATION DIAGNOSIS OF DEFICIENCY OF HYPOXANTHINE PHOSPHORIBOSYL TRANSFERASE IN A MOUSE MODEL FOR LESCH-NYHAN SYNDROME

Male mice embryos deficient in hypoxanthine phosphoribosyl transferase (HPRT), derived from heterozygous (carrier) females and normal males, were diagnosed by biochemical microassay of HPRT activity in a single cell isolated from the eight-cell preimplantation embryo. The sampled embryos were transferred to recipient mothers and examined on the 14th day of gestation to confirm the accuracy of the preimplantation diagnosis. The diagnosis was sufficiently rapid that freezing of the embryos before transfer was not necessary. Of the embryos diagnosed as HPRT negative all 4 that grew into fetuses were correctly identified as HPRT-deficient males.

Biochemical studies on X-chromosome activity in preimplantation mouse embryos.

During early development of female (XX) eutherian mammals, one or the other of the X chromosomes is rendered inactive in all, or most, of the cells of the embryo. This differentiation of the X chromosomes is irreversible, and the adult female is a mosaic with respect to clones of cells with either the maternally-derived or paternally-derived X chromosome inactive. The timing of X-chromosome differentiation has been a subject of considerable interest. Cytogenetic evidence suggests that it occurs around the time of implantation, or at the late blastocyst stage (e.g., see Takagi 1974; Mukherjee 1976). However, other genetic evidence (Gardner and Lyon 1971) suggests that both X chromosomes are active at this stage, at least in the inner cell mass cells of the blastocyst. The subject of X-chromosome inactivation has been extensively reviewed (e.g., see Lyon 1968, 1972, 1974; Eicher 1970; Gartler and Andina 1976; and Monk 1978).