In vivo electroporation of the testis versus transgenic mice model in functional studies of spermatocyte-specific hst70 gene promoter: A comparative study.
To determine whether DNA transfer to mouse testes by in vivo electroporation could be useful method for studying regulatory elements of genes specifically active in spermatocytes first we compared the expression pattern of a construct containing the EGFP reporter gene ligated to a fragment of the heat shock testis-specific hst70 gene promoter, both in testis of transgenic mice and in testis electroporated in vivo. While in transgenic mice the EGFP was expressed in all seminiferous tubules in a cell- and stage-specific manner, in the testes electroporated in vivo only small fraction of cells expressed this marker protein. In order to make a quantitative comparison between the specificity of these two experimental systems we used several vectors containing the CAT gene ligated to fragments of the hst70 gene 5' upstream of DNA sequences which either promoted or did not activate expression of the reporter gene in the testes of transgenic mice. Also, as a reference opposite to spermatogenic cells we examined the expression pattern of the same set of vectors in the rat hepatoma FTO 2B cells. Although electroporated testes retain some spermatocyte-specific features such as the ability to repress promoters which do not contain regulatory elements responsible for testis-specific transcription, several important drawbacks of the method are evident. They include basal activity of constructs which are not transcribed in testes of transgenic mice and low overall transfection efficiency. This may hamper studies in which subtle changes in the expression pattern are under investigation. However, the in vivo electroporation of the testis can be useful for preliminary screening of constructs aimed to study in transgenic mice.
Widłak
Wiesława
W
Department of Tumor Biology, Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Gliwice, Poland.
Scieglińska
Dorota
D
Vydra
Natalia
N
Małusecka
Ewa
E
Krawczyk
Zdzisław
Z
D000818:Animals; D045744:Cell Line, Tumor; D017931:DNA Primers; D018274:Electroporation / Q000379:methods*; D005260:Female; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D051381:Rats; D013090:Spermatocytes / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins