The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alphaalbumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic͞ pituitary system, leading to anovulation.A lpha-fetoprotein (AFP) is a serum glycoprotein produced at high levels during fetal life by the liver and the visceral endoderm of the yolk sac and at lower levels by the developing gastrointestinal tract (1, 2). The protein expressed by the embryo is transferred to the maternal blood circulation, and abnormal levels of embryonic AFP in the maternal serum are indicative of spina bifida or Down's syndrome in the fetus (3, 4). The synthesis of AFP decreases dramatically after birth, and only trace amounts are detected in the adult (2). AFP expression has been shown to be regulated by transcriptional mechanisms involving a relatively large promoter (P1) and three distant enhancers (for reviews see refs. 5 and 6). More recently it has been shown that the first intron of the Afp gene contains an enhancer and an alternative promoter called P2 (7). The genes of the albumin family are linked in the mammalian genomes, and this conserved organization has been proposed to be important for the developmental expression switch of the genes of the family after birth (8). Several hypotheses have been proposed for the physiological function of AFP (for reviews see refs. 6, 9, and 10). Because AFP is synthesized during the cell cycle G 1 and S phases, it has been hypothesized that it affects cell growth (11, 12). The observation that AFP is able to bind estrogen led to the suggestion that AFP plays a role in sexual differentiation of the brain by protecting the fetus from the effects of circulating estrogen (13). In addition to binding estrogen, AFP, like albumin, is able to bind other steroids as well as endogenous and exogenous substances such as fatty acids, bilirubin, and various pharmaceutical agents, suggesting that AFP may play a general transportation role (for review see ref. 14). Moreover, because cellular internalization of the protein has been reported, AFP could also interact with cytoplasmic chaperone proteins that normally escort nuclear receptors or transcription cofactors through the cytoplasm toward organelle interfaces (15, 16). AFP has also been proposed to be one protein that protects the embryo against the maternal immune system, on the basis of the observation that addition of purified AFP into the culture of splenic or lymph node mononuclear cells exerts a suppressive effect on ant...
The use of PCR-amplified DNA-fragments is a classical approach to generate recombinant DNA. To facilitate the cloning of PCR products, we have constructed two new pKIL vectors that allow selection of recombinants. The multiple cloning sites (MCS) of these plasmids contain two adjacent Aspel sites and a unique HindII site. Cleavage of these vectors with Aspel produce linearized molecules with a single thymidine nucleotide at the 3' ends allowing TA cloning of Taq-amplified fragments. On the other hand, cleavage with HindII can be used for the cloning of blunt-ended PCR products generated by other DNA polymerases. The LacZ alpha-CcdB fusion protein produced by these plasmids has retained both the CcdB killer activity and the ability to alpha-complement the truncated LacZ delta M15. This bifunctionality allowed us to show that small PCR products (< 1000 bp) that do not disrupt lacZ alpha efficiently do inactivate CcdB, which demonstrates that the CcdB-based selection is well adapted for cloning of PCR products, especially for small size fragments.
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