Limits...
Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

Schoonbroodt S, Frans N, DeSouza M, Eren R, Priel S, Brosh N, Ben-Porath J, Zauberman A, Ilan E, Dagan S, Cohen EH, Hoogenboom HR, Ladner RC, Hoet RM - Nucleic Acids Res. (2005)

Bottom Line: DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires.Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family.We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

View Article: PubMed Central - PubMed

Affiliation: Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.

ABSTRACT
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Show MeSH
VH family distribution analysis. (A) Frequency of VH family use in healthy donor samples compared with Trimera mice samples (RACE-derived and ONCL-cleaved samples). VH family distribution was compiled for the healthy donor samples from the 265 VH described by de Wildt et al. (32), was studied in 1254 collected heavy chain sequences (VH database) (R. C. Ladner, unpublished data) in 115 clones of RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. (B) Use of human germ-line V-gene segments. Frequencies of use of human VH segments was compiled from the 292 rearranged VH genes in the database described by Tomlinson et al. (33), observed in 115 clones of Trimera mice-amplified and RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. Frequencies (f) are plotted as % of total.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1131936&req=5

fig4: VH family distribution analysis. (A) Frequency of VH family use in healthy donor samples compared with Trimera mice samples (RACE-derived and ONCL-cleaved samples). VH family distribution was compiled for the healthy donor samples from the 265 VH described by de Wildt et al. (32), was studied in 1254 collected heavy chain sequences (VH database) (R. C. Ladner, unpublished data) in 115 clones of RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. (B) Use of human germ-line V-gene segments. Frequencies of use of human VH segments was compiled from the 292 rearranged VH genes in the database described by Tomlinson et al. (33), observed in 115 clones of Trimera mice-amplified and RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. Frequencies (f) are plotted as % of total.

Mentions: To validate our technology, we first studied the distribution of the different VH families in our final library. As a reference, we took the frequency of the VH families in the IgG repertoire of healthy donors described by de Wildt et al. (32), where the authors showed that the VH 1, 3 and 4 families are the most commonly represented in PBLs. We initially determined the sequences of the heavy chains of 35 clones randomly picked from our library. The sequences were aligned with the germ-line VH gene database in order to determine their VH family affiliation. We observed that 17% are representative for family VH 1, 6% for VH 2, 9% for VH 3, 51% for VH 4, 14% for VH 5 and 3% for VH 6. When compared with the distribution in healthy donors, our library had an over-representation of family 4, while family 3 is surprisingly under-represented, suggesting biases induced either through ONCL technology or caused by the modification of the human PBL expression in the Trimera mouse system. In order to understand the origin of this uncommon VH distribution, we determined the sequences of 73 additional heavy chains of the ONCL library together with 115 heavy chains of the amplified V genes before the ONCL cleavage and aligned them to the germ-line VH gene database. As depicted in Figure 4A, the VH distributions observed before (RACE-derived clones) and after the ONCL cleavage are comparable, demonstrating that the VH distribution we observed is not the consequence of our ONCL technology but most probably arose through the Trimera mouse system or the C.albicans infection. Furthermore, as shown in Figure 4B, some VH gene segments (4-61, 4-39 and 4-59) seem to be over-represented when compared with 292 VH sequences collected by Tomlinson et al. (33), showing that the pattern of usage of the segments in Trimera mice spleens (or in C.albicans infected patient PBLs) appears to differ from the usage in healthy donor samples.


Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

Schoonbroodt S, Frans N, DeSouza M, Eren R, Priel S, Brosh N, Ben-Porath J, Zauberman A, Ilan E, Dagan S, Cohen EH, Hoogenboom HR, Ladner RC, Hoet RM - Nucleic Acids Res. (2005)

VH family distribution analysis. (A) Frequency of VH family use in healthy donor samples compared with Trimera mice samples (RACE-derived and ONCL-cleaved samples). VH family distribution was compiled for the healthy donor samples from the 265 VH described by de Wildt et al. (32), was studied in 1254 collected heavy chain sequences (VH database) (R. C. Ladner, unpublished data) in 115 clones of RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. (B) Use of human germ-line V-gene segments. Frequencies of use of human VH segments was compiled from the 292 rearranged VH genes in the database described by Tomlinson et al. (33), observed in 115 clones of Trimera mice-amplified and RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. Frequencies (f) are plotted as % of total.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1131936&req=5

fig4: VH family distribution analysis. (A) Frequency of VH family use in healthy donor samples compared with Trimera mice samples (RACE-derived and ONCL-cleaved samples). VH family distribution was compiled for the healthy donor samples from the 265 VH described by de Wildt et al. (32), was studied in 1254 collected heavy chain sequences (VH database) (R. C. Ladner, unpublished data) in 115 clones of RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. (B) Use of human germ-line V-gene segments. Frequencies of use of human VH segments was compiled from the 292 rearranged VH genes in the database described by Tomlinson et al. (33), observed in 115 clones of Trimera mice-amplified and RACE-derived VH genes, and in 73 clones of the VH repertoire from the immune library. Frequencies (f) are plotted as % of total.
Mentions: To validate our technology, we first studied the distribution of the different VH families in our final library. As a reference, we took the frequency of the VH families in the IgG repertoire of healthy donors described by de Wildt et al. (32), where the authors showed that the VH 1, 3 and 4 families are the most commonly represented in PBLs. We initially determined the sequences of the heavy chains of 35 clones randomly picked from our library. The sequences were aligned with the germ-line VH gene database in order to determine their VH family affiliation. We observed that 17% are representative for family VH 1, 6% for VH 2, 9% for VH 3, 51% for VH 4, 14% for VH 5 and 3% for VH 6. When compared with the distribution in healthy donors, our library had an over-representation of family 4, while family 3 is surprisingly under-represented, suggesting biases induced either through ONCL technology or caused by the modification of the human PBL expression in the Trimera mouse system. In order to understand the origin of this uncommon VH distribution, we determined the sequences of 73 additional heavy chains of the ONCL library together with 115 heavy chains of the amplified V genes before the ONCL cleavage and aligned them to the germ-line VH gene database. As depicted in Figure 4A, the VH distributions observed before (RACE-derived clones) and after the ONCL cleavage are comparable, demonstrating that the VH distribution we observed is not the consequence of our ONCL technology but most probably arose through the Trimera mouse system or the C.albicans infection. Furthermore, as shown in Figure 4B, some VH gene segments (4-61, 4-39 and 4-59) seem to be over-represented when compared with 292 VH sequences collected by Tomlinson et al. (33), showing that the pattern of usage of the segments in Trimera mice spleens (or in C.albicans infected patient PBLs) appears to differ from the usage in healthy donor samples.

Bottom Line: DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires.Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family.We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

View Article: PubMed Central - PubMed

Affiliation: Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.

ABSTRACT
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Show MeSH