Kenneth Simkiss

Transgenic Birds from Transformed Primordial Germ Cells

Primordial germ cells (PGCS) are the progenitor cells for the gametes. They can be obtained from avian embryos by dissociating the germinal crescent region, where they accumulate, or by sampling the blood at the time of their migration to the gonad, PGCS were obtained from these sources and transfected with defective retroviruses. These manipulated cells were injected into recipient embryos to form chimaeras which grew to sexual maturity and produced offspring, some of which contained the foreign DNA. This is the first example of the direct use of PGCS to produce transgenic offspring

Explanted embryo culture: In vitro and in ovo techniques for domestic fowl

Abstract 1. Embryos of the domestic fowl (72 h old) have been explanted into shell‐less cultures or ‘surrogate’ eggshells, in order to investigate the possibility of rearing these embryos to hatching. 2. Rocking embryo cultures during the first half of incubation enhanced embryo growth. 3. Embryos explanted into ‘surrogate’ eggshells of either other individuals or other species have been successfully ‘hatched’. 4. A normal chorioallantois is formed in these surrogate eggshells. This enables a functional albumen sac to form and eggshell resorption to be achieved. 5. Embryos grown in ‘surrogate’ eggshells are slightly smaller than controls but otherwise normal. 6. The technique provides opportunities for genetic engineering experiments.

Bone mineral : evidence for hydroxy groups by inelastic neutron scattering

Inelastic neutron scattering (INS) spectroscopy which is especially sensitive to vibrations of groups with hydrogen has been used to resolve the extent of substitutions at the hydroxy group site in bone. Bone is a complex material consisting of a mineral apatite (Ca5(PO4)3 OH), in an extracellular protein matrix. Many aspects concerning the composition of the mineral are controversial. It has been suggested that there is complete substitution by carbonate at the hydroxy site. Bands assigned to hydroxy vibrations are often not resolved in the infrared and Raman spectra. We show, by INS, that in ox femur bone 40–50% of the hydroxy groups are not substituted. The inelastic neutron scattering experiment opens up new opportunities to monitor changes in bone composition which are important in bone ageing and some pathological conditions.

STERILISATION OF AVIAN EMBRYOS WITH BUSULPHAN

The chemosterilant drug busulphan (1, 4-butanediol dimethane sulphonate) has been applied to embryos of the domestic fowl at doses of from 1 to 500 micrograms after 48 hours incubation. Direct application caused a number of teratological effects. These could be avoided by injecting the drug in sesame oil directly into the yolk. Embryos treated in this way could be sterilised with an efficiency of over 95 per cent with minimal side effects to the young embryo. The technique provides a method for destroying primordial germ cells that will facilitate studies of germ cell/stroma interactions in the gonad and facilitate the production of transgenic birds.

Transfer of primordial germ cell DNA between embryos

SummaryPrimordial germ cells with a retroviral DNA have been transferred to recipient fowl embryos without this marker. The foreign DNA was subsequently identified in the gonads of these birds. This suggests that primordial germ cells may be a useful route for introducing DNA into transgenic birds.

Sterilising embryos for transgenic chimaeras

1. Experiments were undertaken to attempt to sterilise fowl embryos with ultraviolet light. Such sterilised embryos would be useful as recipients of genetically manipulated germ cells. 2. The germinal crescents of embryos were exposed to a calibrated UV source at stages 4 and 8 to 10 of incubation for 30 s, 3 min and 10 min. Teratological and sterility effects were studied at periods up to 6 d of incubation. 3. Simply exposing embryos by opening the shell produced a number of abnormalities and mortalities. These decreased with the age of the embryo but increased with the dosage of irradiation. 4. Although there was abundant evidence for UV-induced cell damage, the sterility of the embryos was usually less than 75%.

Ballistic transfection of avian primordial germ cellin ovo

In the fowl the primordial germ cells accumulate in the germinal crescent to the anterior of the two-day embryo. A simple ballistic device has been used to fire tungsten particles (mean diameter 1.5 μm) into this region. By coating these projectiles with vector DNA it is possible to transfect these cells. Hatchlings produced by this technique were raised to sexual maturity and shown to contain the foreign DNA in their sperm. G1 offspring containing this DNA were also produced in roughly 20% of these cockerels. In the majority of cases the vector DNA disappeared from the G1 generation as they matured suggesting that in these cases it had been transmitted episomally.

The ultrastructure and metal-containing inclusions of mature cell types in the hepatopancreas of a crayfish.

The ultrastructure of the hepatopancreas of the crayfish Austropotamobius pallipes (Lereboullet) is described in relation to its function in digestion. X-ray microprobe analyses of the cells of this tissue have been obtained after using a variety of fixatives and plasma oxygen etching. The distribution of metals in the digestive cells is considered in relation to the polarization of the cells and their ability to absorb materials across the apical and basal cell membranes.

Corrosion of Intracellular Granules and Cell Death

The snail Helix aspersa has large numbers of calcium cells in its hepatopancreas which contain membrane-bound intracellular granules of CaMgP2O7. These inorganic deposits are the sites of accumulation of a wide variety of cations and act as a detoxification mechanism that traps a number of dietary metals once they have entered the animal. This study concentrates on the mechanism of incorporation of manganese ions into these deposits by using electron microscopy, ultra-low-angle X-ray scattering and extended X-ray absorption spectroscopy. The results show that manganese ions cause a localized corrosion of these intracellular granules with a consequent release of calcium ions. This release appears to overwhelm the calcium-regulatory mechanisms of these cells and leads to their death and subsequent shedding into the lumen of the alimentary tract. The concept of intracellular corrosion is a novel interpretation of a previously unsuspected biochemical lesion.

FEMALE CHROMOSOMES IN COCKEREL EJACULATES

We describe a polymerase chain reaction which amplifies part of the Eco RI repeat unit of the fowl W chromosome. The resulting 447 bp fragment enables DNA from female birds to be identified. The composition of this DNA is confirmed by a nested polymerase chain reaction which specifically amplifies a known internal 263 bp region in this fragment. Using this technique it is possible to follow the fate of female cells in male germline chimaeras. The polymerase chain reaction fragment can be traced in cells of the embryonic and hatchling gonad and in adult sperm implying that cells containing the W chromosome are capable of being processed through the avian testis.