An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation

Background: Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals. Methods: A solution of busulfan, ranging from 25 μg/20 μl to 100 μg/20 μl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 μl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring. Results: A dose of 75 μg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods. Conclusions: We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation

Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique

A non-surgical approach for male germ cell mediated gene transmission through transgenesis

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers

Role of H-bond interactions in the protonation and wavelength regulation in retinal schiff base chromophores

Absorption spectral studies of all-trans-N-retinylidene-n-butclamine (1, all-trans-N-retinylidene-tryptamine (2) in various media (heptane, methanol, cellulose, cellulose acetate, ethylene glycol, polyethylene glycol, polyethylene, and polyvinyl alcohol) and in presence of various protonating agents (chloroacetic acid, 2-chloropropionic acid, perchloric acid) suggest that H-bond interactions play important role in regulating the absorption spectrum of retinylidene Schiff base chromophore. The results are discussed in terms of the characteristic absorption spectral properties of retinal based photoreceptor proteins

Bathochromicity of retinal schiff bases in cellulose matrix

Schiff bases of all-trans-retinal (formed with n-butylamine, tryptamine and β-naphthylamine) and of benzaldehyde, trans-cinnamaldehyde and all-trans-retinal with aniline exhibit an appreciable red shift in their UV-visible maxima on intercalation in cellulose matrix relative to their absorption in solution in the absence of acid. Treatment of these model compounds with trichloroacetic acid in solution gives the corresponding protonated salts. The red shift due to the cellulose environment is, however, less than the red shift in acid solutions. However, an exception is all-trans-N-retinylidenetryptamine for which the red shift in cellulose is quite close to the corresponding value for the protonated salt in heptane and methanol. N-Benzalideneaniline and trans-N-cinnamalideneaniline, with shorter polyenic moieties, tend to show a greater bathochromic shift in cellulose. all-trans-N-Retinylidene-n-butylamine, all-trans-N-retinylidenetryptamine and all-trans-N-retinylidene-β-naphthylamine show a reduced bathochromic shift when intercalated in cellulose pretreated with a base such as n-butylamine. The chromophore of all-trans-N-retinylidenetryptamine is stabilized by the presence of the indole moiety. These results indicate the importance of hydrogen-bond interactions at the chromophore sites of rhodopsins. A mechanistic proposal for explaining protonation, stability and wavelength regulation in the opsin family of proteins is discussed

Photoactive bacteriorhodopsin variants

Bacteriorhodopsin variants were generated by UV mutagenesis. The protein variants thus engineered were characterized for their UV-visible absorption, light induced proton release activity and photocycle kinetics (BR→M)

Surgical approach to Parapharyngeal Tumours - Our Experience

Introduction The complex anatomy of the parapharyngeal space, the surrounding vital structures and late presentation of the tumours affecting this space pose difficult surgical challenge to every otolaryngologist. The study gives an overview of the experience in managing parapharyngeal tumours in a tertiary care setting. Materials And Methods Fifteen patients with parapharyngeal tumours were treated surgically in a medical college hospital in Kolkata in the three year study period. Surgical approaches were chosen, considering the size, site, extent and histology of the tumours. Results Most of the patients (33.33%) were from 20-30 years age group. 60% were females. The most common presenting feature was neck swelling. 80% of the cases were benign.The most common histologic variant was Schwannoma. The tumours were accessed through Trans-cervical, Trans-cervical Trans-parotid or Trans-cervical with Midline Mandibulotomy approaches. The most common post-operative complication was wound dehiscence. Discussion The most common presentation of a parapharyngeal mass was neck swelling mainly behind the angle of mandible (86.67%). Younger patients in their third decade of life were found to be affected more. Gadolinium enhanced MRI was found to be the most important investigation in parapharyngeal tumours. Complications were minimal. Conclusion A conservative trans-cervical approach was found to be feasible and effective in majority of the cases over radical ones, which may be required in malignancies and skull-base involvement