A murine a-lactalbumin-null allele was created by homologous recombination in ES cells and introduced in the mouse germ-line. Mice homozygous for this allele produced a highly viscous milk, devoid of lactose, with higher protein and fat contents, that pups were unable to suck out. No other obvious phenotype was observed. Thus, a-lactalbumin is the sole protein able to induce lactose synthesis in vivo, which emphasizes its key function in the lactation process.
Stinnakre, M.G., Vilotte, J.L., Soulier, S. and Mercier, J.C. (1994) Proc. Natl. Acad. Sci. USA 91, 6544-6548.
Stacey, A., Schnieke, A., Kerr, M., Scott, A., McKee, C., Cottingham, I., Binas, B., Wilde, C. and Colman, A. (1995) Proc. Natl. Acad. Sci. USA 92, 2835-2839.
Note: mini-review on the alpha-lactalbumin gene
a-lactalbumin and k-casein are the only two major milk proteins that have been found in every milk studied so far. It probably relates to the important roles attributed to these two proteins: formation of micelles for k-casein and induction of lactose synthesis, one of the key osmotic regulator of milk secretion, for a-lactalbumin. However, that only these two proteins can perform such functions in vivo remained to be demonstrated. Other biological functions were attributed to a-lactalbumin, such as inhibition of epithelial cell growth, induction of apoptosis and a cell lytic activity. A reduction in the lactose content of the milk could be beneficial both by increasing the overall milk concentration and providing food ressources for lactose intolerant people. To assess the potential of manipulating milk composition by modifying a-lactalbumin gene expression, ES cells and gene targeting were used to introduce into the mouse germ line a null allele at this locus.
Gene Targeting Vector
The gene-targeting vector contained 5 kb of the mouse a-lactalbumin BALB/c 5' flanking
sequence (-5.56 to -0.56), an HSV-neomycin-resistance cassette, a 2.6 kb fragment
encompassing the transcription unit of the gene downsteam from exon 2 and 1 kb of 3'
flanking sequence and a HSV-tymidine kinase cassette. The two cassettes were kindly
provided by Dr. P. Simons (now at The Royal Free Hospital School of Medecine, London, UK).
Thus, following gene targeting, the mutated gene lacked the first 560 bp of its 5'
flanking sequence and part of its transcription unit 5' to intron 2.
Mouse Strain
Gene targeting was performed in 129-derived D3 ES cells (kind gift from Dr. Kemler,
Max-Planck-Institut f,r Immunbiologie, Freiburg, Germany). Injection of cells and further
mating were performed using a C57BL/6 genetic background.
Phenotype
Following establishment of the lines, the mutated gene was transmitted in a Mendelian
fashion. However, females homozygous for the mutated gene (-/-) were unable to foster pups
that could not suck out their milk. Milk from -/- mice, compared with that of +/+ mice,
lacked of a-lactalbumin and detectable lactose, had higher protein and fat contents and
viscosity; milk from +/- mice had an intermediate composition (see table). SDS-PAGE of
milk proteins and Northern analysis demonstrated that all major, but a-lactalbumin, milk
proteins were secreted and their respective genes equally transcribed in these three types
of mice. As compared with +/+ or +/- mice, the extracellular spaces of the mammary gland
from -/- mice were found to be invaded by fat globules and their epithelial cells had a
reduced volume of cytoplasm, secretory vesicles and Golgi apparatus
INRA-CRJ
78352 Jouy-en-Josas, Cedex
France
Phone: (33 1) 34 65 25 76
Fax: (33 1) 34 65 24 78
e-mail: vilotte@biotec.jouy.inra.fr
contributed July 1996