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Monoclonal antibodies (MAbs) have been utilized as research tools, as diagnostic reagents, and for antibody medicine. The preparation of MAbs involves laborious processes and normally takes months. Here we describe an ex vivo B cell-based antibody display system called the ADLib (Autonomously Diversifying Library) system, which enables us to select chicken B cell clones producing antibody against antigens of interest in a couple of weeks. The ADLib system is applicable to self- or highly conserved antigens, polysaccharide chains, peptides, and haptens.

Transthyretin (TTR) related familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid deposits in the peripheral nerves and other organs. Liver transplantation has been utilized as therapy for FAP, because the variant TTR is predominantly synthesized by the liver, but this therapy is associated with several problems. Since we need to develop a new treatment, we examined the feasibility of gene therapy in FAP to prevent the production of variant TTR in the liver by using single-stranded oligonucleotides (SSOs). To examine in vitro and in vivo conversion of the TTR gene by SSOs embedded in atelocollagen, we used HepG2 cells and liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the mouse TTR V30M gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. The level of gene conversion was approximately 11% and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Therefore, gene therapy via this method may be a promising alternative to liver transplantation for treatment of FAP.

Two steroid 21-hydroxylase genes (CYP21A and CYP21B) alternate in tandem with two genes for the fourth component of complement (C4A and C4B) on the short arm of chromosome 6 between the loci of HLA-B and HLA-DR. The CYP21B gene encodes an adrenal microsomal cytochrome P-450, which is specific for steroid 21-hydroxylation (P450c21). A defect of this protein would cause 21-hydroxylase deficiency, which is an autosomal recessive disease and is the most common cause of congenital adrenal hyperplasia (CAH). On the other hand, the CYP21A gene, which is homologous to the CYP21B gene up to 98% in the nucleotide sequences, is a pseudogene due to several mutations in the coding region. One of the mutations is a C----T change leading a termination codon, TAG, in the 8th exon. 1) I cloned a CYP21B gene from a patient homozygous for HLA-Bw75-DRw9 by descent. I found a C----T change in the 8th exon of the CYP21B gene. This mutation would prevent a synthesis of 21-hydroxylase and was thought to be a crucial change to cause CAH in this patient. Because there was no apparent gross change in the organization of the C4-CYP21 region and this mutation is usually found in the CYP21A pseudogene, it seemed that a gene conversion-like event transferred the mutation from the CYP21A gene to the CYP21B gene. 2) A population study on the organization of C4-CYP21 region revealed that a reciprocal change, i.e. a T----C change in the 8th exon of the CYP21A gene, was observed in two HLA haplotypes, HLA-B44-DRw13 and HLA-Bw46-DRw8 haplotypes in Japanese population. The reciprocal changes also may be considered as a result of gene conversion-like events.