Isolation and some effects of functional, low-phenylalanine kappa-casein expressed in the milk of transgenic rabbits.

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.

Characterization, chromosomal assignment, and role of LIFR in early embryogenesis and stem cell establishment of rabbits.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine with an important role during early embryonic development, implantation, and as an inhibitor of murine embryonic stem cell differentiation. It exerts its effects by binding to the leukemia inhibitory factor receptor, a heterodimer of two transmembrane proteins, the specific leukemia inhibitory factor receptor subunit, and the common gp130. A partial cDNA clone coding for the membrane-bound form of the specific rabbit leukemia inhibitory factor receptor was isolated from the genital ridge of 13.5 days postcoitum fetus. Fluorescent in situ hybridization analysis revealed that the rabbit leukemia inhibitory factor receptor gene is located on chromosome OCU11p11.1. It has been shown that the membrane-bound rabbit leukemia inhibitory factor receptor mRNA is expressed during embryo implantation but not at earlier developmental stages. Rabbit embryonic stem cell-like line establishment is improved in the presence of LIF, and those cells express both leukemia inhibitory factor and its receptor. The withdrawal of leukemia inhibitory factor results the differentiation of embryonic stem cell-like cells to beating myocardial-like cells. Our findings suggest that the self-renewal mechanism is similar in mouse and rabbit embryonic stem cells, and expands our knowledge on the role of the LIF-LIFR signal pathway in early rabbit embryogenesis and rabbit embryonic stem cell establishment.