Preimplantation diagnosis of a human beta-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.
The preimplantation diagnosis of a HbSA-globin transgene in biopsied trophectoderm cells and blastomeres in embryos using a transgenic mouse model for the trait of human sickle-cell anaemia has been undertaken. A sensitive procedure was developed for the amplification of the human beta-globin gene sequence flanking the sickle mutation. Polymerase chain reaction (PCR) assays were undertaken on one to five biopsied trophectoderm cells and isolated blastomeres of the preimplantation mouse embryo. After biopsy the blastocysts were cultured whilst the cells were analysed for the presence of the transgene, and a high proportion (82-91%) were viable as assessed by the presence of a blastocoele cavity within a 5-h period. The majority of the biopsied cultured blastocysts were frozen and used to confirm the diagnosis; 90 biopsied cultured blastocysts were transferred to pseudopregnant recipients and 34% established pregnancy. Material from day 13.5 post-coitum fetuses was also used to confirm the original diagnosis. The time (4-5 h) required to carry out the analysis obviates a need for extended culture or cryopreservation of the biopsied embryo. In individual experiments under optimal conditions, the presence of the transgene in biopsied cells was detected with 100% accuracy, and the PCR analysis was sensitive at the 1-cell level. The overall success rate of diagnosis and confirmation of the presence or absence of the human beta-globin sequence in the biopsied embryo was 70%. Over the entire experimental period (14 months) DNA contamination from a variety of sources did occasionally occur; the methods used to overcome this problem are discussed.
MRC Experimental Embryology and Teratology Unit, St George's Hospital Medical School, London, UK.
S A
SA
Sheardown
I
I
Findlay
A
A
Turner
D
D
Greaves
V N
VN
Bolton
M
M
Mitchell
D M
DM
Layton
A L
AL
Muggleton-Harris
D000755:Anemia, Sickle Cell / Q000175:diagnosis*; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D047108:Embryonic Development; D005260:Female; D006451:Hemoglobin, Sickle / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011247:Pregnancy; D011296:Prenatal Diagnosis; D012680:Sensitivity and Specificity
D006451:Hemoglobin, Sickle; D015345:Oligonucleotide Probes; D004247:DNA