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. 2002 Apr;3(4):367-72.
doi: 10.1093/embo-reports/kvf073.

Use of stepwise subtraction to comprehensively isolate mouse genes whose transcription is up-regulated during spermiogenesis

Takayuki Fujii  1 , Kazutoshi Tamura, Kumiko Masai, Hiromitsu Tanaka, Yoshitake Nishimune, Hiroshi Nojima
Affiliations

Use of stepwise subtraction to comprehensively isolate mouse genes whose transcription is up-regulated during spermiogenesis

Takayuki Fujii et al. EMBO Rep. 2002 Apr.

Abstract

We report the isolation of 153 mouse genes whose expression is dramatically up-regulated during spermiogenesis. We used a novel variation of the subtractive hybridization technique called stepwise subtraction, wherein the subtraction process is systematically repeated in a stepwise manner. We named the genes thus identified as TISP genes (transcript induced in spermiogenesis). The transcription of 80 of these TISP genes is almost completely specific to the testis. This transcription is abruptly turned on after 17 days of age, when the mice enter puberty and spermiogenesis is initiated. Considering that the most advanced cells present at these stages of spermatogenesis are the spermatids, it is likely that we could isolate most of the spermatid-specific genes. DNA sequencing revealed that about half the TISP genes are novel and uncharacterized genes, confirming the utility of the stepwise subtraction approach for gene discovery.

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Figures

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Fig. 1. A stepwise subtraction strategy for comprehensively isolating genes whose transcription is induced during spermiogenesis. (A) The TISP cDNA clones obtained by stepwise subtraction can be classified into three types according to the pattern of band intensity in northern blots with juvenile and adult testicular RNA. A northern blot with radiolabeled β-actin cDNA is shown as a loading control. See text for details. (B) The effect of the stepwise subtractions on the total harvest of TISP clones from the cDNA library can be monitored by plotting the total number of non-redundant cDNA clones analyzed after each stage (abscissa) against the number of TISP cDNA clones that were isolated in each stage (ordinate). The shapes of the harvest curve that might be drawn without performing the stepwise subtractions are shown by broken lines. (C) The actual harvest curve for each type of TISP cDNA clone resulting from performing the five stepwise subtractions. The isolated TISP clones are divided into the three classes. Numbers shown at each node of the curves signify the total number of TISP cDNA clones isolated by the end of each subtraction step. The nodes represent the five hybridizations that were performed on the cDNA library.
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Fig. 2. Expression of each Type 1 TISP cDNA clone. Northern blot analysis was performed by hybridizing the radiolabeled cDNA insert of each TISP clone to RNA from various murine tissues, from the testes of four kinds of mutant mice or from the testes of mice of varying ages. B, brain; H, heart; I, intestine; K, kidney; Li, liver; Lu, lung; M, skeletal muscle; O, ovary; S, spleen; T, testis; C, cryptorchid testis; j, testis of jsd/jsd mouse at 3 months of age; sl, testis of Sl17H/Sl17H mouse; w, testis of W/WV mouse. The number shown at the right of each blot denotes the size of the mRNA as assessed by the RNA size marker run in a parallel lane of each agarose gel (not shown). A northern blot with radiolabeled β-actin cDNA is shown as a loading control. The accession number for each TISP cDNA sequence (registered in the DDBJ data bank) is shown beside each northern blot.
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