| Autor: |
Majumdar, Subeer S., Usmani, Abul, Dhup, Suveera, Sarkar, Hironmoy, Batta, Suryaprakash R., Vimal, Manoj, Ganguli, Nirmalya |
| Zdroj: |
Biology of Reproduction; July 2009, Vol. 81 Issue: 1, Number 1 Supplement 1 p96-96, 1p |
| Abstrakt: |
With the availability of the human and mouse genomes, there is an increasing need for developing variety of transgenic mouse models. Presently used technique for making transgenic animals involves pronuclear DNA microinjection by embryo manipulation. This is cumbersome, expensive, time consuming and it requires large number of females for super-ovulation which are then killed. We have recently established a new technique for generating transgenic mice by electroporation of the transgene in spermatogonial cells of testis. This technique has some caveats. It requires surgical exposure of the testis which may remain a potential site of infection. Inappropriate survival surgery may also lead to impotency. Moreover, surgical procedure may not be suitable for large animals under farm condition. Hence, the technique may remain unpopular in spite of several advantages. Development of a procedure avoiding surgeries may not only be ethically superior but also pave the way for frank use of this technique in small and large animals. With this objective, we have developed a non invasive procedure for integration of the foreign DNA in repopulating spermatogonial cells of the testis. For this purpose, the linearized DNA(20-25μl of 0.5 μg/μl) containing promoter and gene of interest was injected aseptically by Hamilton syringe into the testis of anesthetized mouse. Several conditions for electroporation were tested; electroporation using 8 square 60V electric pulses in alternating direction with a time constant of 50-60 milli second and an inter-pulse interval of ~1 second worked best. The whole procedure can be accomplished within 10 minutes. Testis of a mouse expressed green fluorescence, 50 days after eletroporation with a construct containing EGFPgene cloned downstream of chicken β-actin promoter (pCX-EGFP) confirming genomic integration of the transgene. Since a cycle of spermatogenesis in mice is completed in about 35 days, the electroporated males were mated after 35 days of electroporation. This was done to ensure that the progenies bearing transgene must result from sperm generated from the repopulating spermatogonial cells which were successfully transfected at the time of electroporation. Analysis of transgene by PCR of genomic DNA obtained from tail tips of progeny generated by mouse electroporated with i) pCX-EGFP, ii) the fusion gene of full-length fragment of Scaffold/Matrix attachment region binding protein1(Smar1) and EGFPcloned downstream of Cytomegalovirus immediate early promoter/enhancer (Smar1-EGFP) and iii) human insulin-like growth factor binding protein-6 (IGFBP-6)cloned downstream of a brain specific promoter of glial fibrillary acidic protein (Gfap-IGFBP6) validated that our procedure resulted into successful production of transgenic mice with all these constructs. More than 50% of the pups born were transgenic. EGFP was expressed by the liver of mouse transgenic for Smar1-EGFP. As expected, GFAP expression by astroglial cells was diminished in mouse (F3 generation) transgenic for Gfap-IGFBP6because presence of IGFBP6 in the milieu of brain is known to sequester IGF2 which is essential for healthy development of astroglial cells. This is the first report about development of a non-surgical, rapid and ethically superior deathless technique for making transgenic animals. It generates a potential scope for its widespread use in divulging functions of genes and in developing animal models of pathophysiological importance.(platform) |
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