Characterizing transgene inheritance.

The analysis of transgene inheritance is an important step in the molecular and genetic characterization of transgenes. In this manuscript, two approaches to characterize the inheritance of transgenes are described. The first approach is based on the expression of the transgene phenotype and the second is based on the analysis of transgene DNA. Instructions on how to make crosses and develop breeding populations are outlined and the importance of these breeding populations in the analysis of transgene inheritance is explained. The number of individuals needed to determine segregation ratios and the statistic used to test these ratios are described. Examples of inheritance patterns that deviate from known expectations are provided and the possible causes of these deviations are discussed.

Transgenic maize endosperm containing a milk protein has improved amino acid balance.

In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed transgenic maize lines that produce the milk protein alpha-lactalbumin in the endosperm. Lines in which the transgene was inherited as a single dominant genetic locus were identified. Sibling kernels with or without the transgene were compared to determine the effect of the transgene on kernel traits in lines selected for their high content of alpha-lactalbumin. Total protein content in endosperm from transgene positive kernels was not significantly different from total protein content in endosperm from transgene negative kernels in three out of four comparisons, whereas the lysine content of the lines examined was 29-47% greater in endosperm from transgene positive kernels. The content of some other amino acids was changed to a lesser extent. Taken together, these changes resulted in the transgenic endosperms having an improved amino acid balance relative to non-transgenic endosperms produced on the same ear. Kernel appearance, weight, density and zein content did not exhibit substantial differences in kernels expressing the transgene when compared to non-expressing siblings. Assessment of the antigenicity and impacts on animal health will be required in order to determine the overall value of this technology.

Expression of a synthetic porcine alpha-lactalbumin gene in the kernels of transgenic maize.

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine alpha-lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3' terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The alpha-lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine alpha-lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5' end of the synthetic porcine alpha-lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine alpha-lactalbumin accumulation in transgenic maize kernels.