Michael Gurevich

Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

Abstract Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

Factors affecting the concentration of testosterone in poultry litter

Abstract Poultry litter used as cattle feed can contain high levels of testosterone (>400 ng/g) and estrogen (>300 ng/g) which can interfere with reproduction. However such high levels of testosterone are not usually found in broiler chicken litter (< 6% of samples). We tested the following parameters to determine what factors could be responsible for highly elevated concentrations of testosterone: sex, age, fermentation. It was found that (1) there is no difference in testosterone content in poultry litter obtained from male (133±12 ng/g) or female broilers (133±13 ng/g); (2) poultry litter from adult roosters have unacceptably high levels of testosterone (670±95 ng/g) and layers have high levels of estrogens (533±50 ng/g); (3) although fermentation of poultry manure generally lowers steroidal hormone content, if antibiotics are added to the incubation, there was a a five‐fold increase in radioimmunoassayable testosterone content (controls 94±6 ng/g vrs treated 578±80 ng/g).

Induction of phytoestrogen production in Medicago sativa leaves by irrigation with sewage water

The phytoestrogenic activity of alfalfa irrigated with sewage water was determined by radioreceptor assay (RRA) and high pressure liquid chromatography (HPLC). It was found that irrigation of alfalfa fields increased the phytoestrogenic activity measured in the leaves from 10 ng estrogen equivalents (est. eqv.) to 137 ng est. eqv. g−1, which was principally due to a rise in coumestrol (<10 μg to 60 μg g−1). Similarly, when plants grown in greenhouses under controlled conditions were irrigated with sewage water, the phytoestrogenic content rose from 34 to 48 est. eqv. g−1; (P<0.01; n = 16). Under hydroponic conditions, addition of 1 nM of estrone to the media resulted in a significant rise in phytoestrogen content in the leaves within 48 hr (from 21.2 to 34.5 est. eqv. g−1; P<0.05, n = 10). Alfalfa irrigated with sewage water may therefore have levels of coumestrol (<30 μg g−1) which can affect fertility in cattle. The agents in the sewage water responsible for increased phytoestrogen content of the alfalfa are probably the steroidal estrogens, estradiol and estrone, which are present in sewage in concentrations ranging from 10 to 300 ng l−1.

Sperm-Mediated Gene Transfer: Concepts and Controversies

Description: Sperm-mediated gene transfer (SMGT) represents a novel set of technologies for animal (or in the future, human) genetic modification using the sperm as a vector, as opposed to more traditional established routes such as fertilized eggs or embryonic stem cells. Studies of sperm-mediated gene transfer (SMGT) indicate that sperm cells possess the ability to be utilized as carriers of exogenous genetic sequences, offering the potential of a novel cost-effective route for germline genetic modification. The fate of transgenes borne by sperm cells has been inconsistent, and analysis of offspring from SMGT experiments has shown a mixed picture in terms of genomic integration of the transgene, suggesting an episomal mode of inheritance. Various distinct steps in transgene uptake by the sperm cell have been described or proposed, including a model based upon endogenous reverse transcriptase activity. Although mature sperm cells are naturally protected against uptake of foreign nucleic acid molecules, certain environmental conditions, for example at key times within the reproductive tract, may reduce this protection, suggesting that SMGT may occasionally take place in nature. If correct, this carries profound implications for evolution and human genetic health. This e-book brings together theoretical and empirical reviews from experts in SMGT, providing comprehensive coverage of the major trends, developments and controversies in this novel field. This e-book is intended as a reference for professional researchers in the field of animal genetic modification (transgenesis) as well as teachers, scientists and physicians interested in medical genetics in general and gene therapy in particular.