[Introduction of a new method for mono-specific antibody production by sequential use of recombinant proteins and synthetic peptides [PrIPeP model]]

Objective: This study attempted to generate monospecific antibodies through immunization with recombinant proteins and subsequent purification by synthetic peptides [the PrIPeP model]

Methods: The SRY gene was cloned on a pet-28a vector and the recombinant protein was expressed in the Escherichia coli [E.coli] BL21 strain. The purified antigen was emulsified in Freund's adjuvant and injected into rabbits according to a standard time table. Then, a specific peptide was designed, synthesized, and conjugated to sepharose 4B to generate an affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization, as above. Antisera against the conjugated peptide [Pepantisera] and SRY recombinant protein [Pro-antisera] were evaluated by ELISA and subsequently subjected to the affinity purification column. Sensitivity and specificity of the purified antibodies against SRY recombinant protein as well as negative controls [recombinant HSFY, RBMY, and RPSFY] were assessed by Western blot analysis

Results: Titration by ELISA confirmed proper immunization and specificity of both antigens. Western blot analysis validated the specificity and sensitivity of the IgG class purified antibodies

Conclusion: By applying the PrIPeP model, it is possible to develop antibodies against the native structure of a protein whilst avoiding challenges of peptide-carrier protein conjugation

Stauprimide priming of human embryonic stem cells toward definitive endoderm

In vitro production of a definitive endoderm [DE] is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells [hESCs], researchers have yet to discover universal, efficient and cost-effective protocols. In this experimental study, we have treated hESCs with 200 nM of Stauprimide [Spd] for one day followed by activin A [50 ng/ml; A50] for the next three days [Spd-A50]. In the positive control group, hESCs were treated with Wnt3a [25 ng/ml] and activin A [100 ng/ml] for the first day followed by activin A for the next three days [100 ng/ml; W/A100-A100]. Gene expression analysis showed up regulation of DE-specific marker genes [SOX17, FOXA2 and CXCR4] comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change [p<0.05]. We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd [400 and 800 nM] in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A [25 ng/ml] was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 [IDE1 and IDE2], two previously reported small molecule [SM] inducers of DE, in our protocol [Spd-IDE1/2]. This replacement resulted in the up regulation of visceral endoderm [VE] marker [SOX7] but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs These findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs

Expression analysis of RNA-binding motif gene on Y chromosome [RBMY] protein isoforms in testis tissue and a testicular germ cell cancer-derived cell line [NT2]

Background: RNA-binding motif gene on Y chromosome [RBMY], a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages

Methods: full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms

Results: selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate

Conclusion: the results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Iran. Biomed. J. 17[2]: 54-61, 2013