Gamal E. Osman

Rapid cloning of any rearranged mouse immunoglobulin variable genes

Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologist. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5′ and 3′ universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36–60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny.

FVB/N (H2(q)) mouse is resistant to arthritis induction and exhibits a genomic deletion of T-cell receptor V beta gene segments.

Abstract Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis (CIA) in mice is an autoimmune disease model of rheumatoid arthritis. Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). CD4+ T cells that express the T-cell receptor (TCR) Tcra-V11.1 and/or Tcrb-V8.2 play a key role in the pathogenesis of arthritis in the DBA/1 mouse (H2q). We identified an inbred mouse strain, FVB/NJ (H2q), that is resistant to arthritis induction and exhibits a genomic deletion of certain Tcrb-V gene segments. We report a novel polymerase chain reaction-based method for the rapid identification of new mouse strains that exhibit germline Tcrb-V gene deletions. We mapped for the first time both the 5′ and 3′ breakpoints of the Tcrb-V deletion in the FVB/NJ, SWR, SJL, C57L, and C57BR strains to within 1.1 kilobases. Since there is an association between a particular Tcra-V allele (Tcra-V11.1d) and arthritis susceptibility in H2q mouse strains, we examined the allelic polymorphisms of the Tcra-V11 gene subfamily members between the arthritis-susceptible DBA/1 mouse and the arthritis-resistant FVB/NJ mouse strain. The amino acid sequences of the Tcra-V11.1 alleles differ at two positions (codons 18 and 68). Therefore, the resistance of FVB/NJ mouse to arthritis induction may be due in part to Tcra-V11.1 coding sequence polymorphism and Tcrb-V8.2 gene segment deletion, as we have recently demonstrated in the case of SWR mouse strain.

T-cell receptor vbeta deletion and valpha polymorphism are responsible for the resistance of SWR mouse to arthritis induction.

Abstract Collagen type II-induced arthritis (CIA) develops in susceptible mouse strains after intradermal injections of type II collagen (CII) in complete Freunds adjuvant (CFA). Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). Although the SWR mouse has a susceptible MHC haplotype (H2q), it is resistant to CIA. SWR exhibits at least two known immunological defects: (1) it contains a germline deletion of about 50% of T-cell receptor (TCR) Vβ-chain gene segments, and (2) SWR is deficient in complement component C5. It has been shown that T cells that express TCRVα11.1 and TCRVβ8.2 play a substantial role in the pathogenesis of arthritis in the DBA/1 mouse (H2q). We generated SWR transgenic (tg) mice to determine whether the expression of pathogenic Vα11.1 and/or Vβ8.2 transgenes would confer arthritis susceptibility. Arthritis was induced in the SWR TCRαβ tg mice, but not in SWR TCRβ tg mice. To address the role of Vα11.1 in arthritis susceptibility, we examined the allelic polymorphisms of the Tcra-V11-gene subfamily members between the arthritis susceptible DBA/1 mouse and the arthritis-resistant SWR mouse strain. The amino acid sequences of the Vα11.1 alleles differ at two positions (codons 18 and 68). Accordingly, these two amino acid changes are sufficient to allow the production of pathogenic T cells in SWR mice. This is the first demonstration of the association of a particular Tcra-V allele and arthritis susceptibility in mice.

Characterization of the mouseTcra-V22 gene subfamily

T-cell receptors (Tcrs) of higher organisms play a key role in the specific recognition of self and non-self molecules in the immune system. The large number ofTcr variable (V) genes have been organized intoV gene subfamilies according to their sequence similarity at the nucleotide and amino acid level. We cloned and characterized four new members of theTcra-V22 gene subfamily at the genomic level using a simple and sensitive technique that can rapidly clone members of any multi-member gene family. Sequence analysis reveals that the fourTcra-V22 gene subfamily members have more than 98% sequence similarity in their coding regions, at the nucleotide and amino acid levels. However, the intron between the leader and the coding region varies up to 7% between members of theTcra-V22 gene subfamily. Comparison of the multimemberTcra-V22 gene subfamily with other multi-memberTcra-V gene subfamilies (V2, V8, andV11), shows thatTcra-V22 is unique in that it has multiple members with nearly identical amino acid sequence and which are not inherently pseudogenes. Sequence similarity analysis of theTcra-V22 subfamily with the prototypes of all otherTcra-V subfamilies revealed that theTcra-V22 subfamily has the closest sequence similarity to that ofTcra-V18 (77% at the nucleotide level and 71% at the amino acid level).