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The Makings of a Man

08/15/2014
Jim Kling

When overexpressed in female mice, the SRY gene leads to a male phenotype, but eliminating SRY expression in male mice isn’t easy. Now researchers have accomplished this, but did they find what they expected? Find out...


When researchers presented the first knockout mice made using new TALEN genome engineering technology (1), Shuji Takada at the National Research Institute for Child Health and

SRY knockout mouse (left) and wild type female mouse (right)

Development in Tokyo, Japan saw an opportunity to finally target genes on the Y-chromosome. The sequence of the Y-chromosome is highly repetitive, a characteristic that makes it notoriously difficult to study since the most commonly used method for creating knockout mice relies on homologous recombination, a process that is unpredictable in highly repetitive regions.

TALEN technology relies on a sequence-specific endonuclease comprising a proteobacterial transcription activator-like effector (TALE) and a nuclease domain from FokI restriction endonuclease. When two TALENs bind DNA, the nuclease domain creates a double-stranded break. The resulting cut is repaired by non-homologous end joining, which allows insertion or deletion at the cut site.

The TALEN method is compatible with DNA-binding domains as short as 45-65 residues in length, making it ideal for working with the SRY gene. And embryonic stem (ES) cells aren’t required for creating knockout animals since TALENs can be injected directly into fertilized oocytes.

“We showed that even a Y-linked gene can be knocked out without using ES cells in a short period of time. We believe TALEN enables us to make knockout mice for genes which were difficult to disrupt,” Takada wrote in an email.

Using TALEN technology, Takada’s group created XY mice lacking the SRY gene (2). These mice had female genitalia both externally and internally and blood testosterone levels similar to those found in females. The animals went through estrus and copulated as females, but were either infertile or had reduced fertility.

Further examination revealed that the animals’ ovaries had a reduced number of egg precursors and also contained luteinized un-ruptured follicles (LUFs) where the egg hadn’t been released. Takada and his group are interested in this characteristic since infertile women often have a higher incidence of LUFs, indicating that SRY knockout mice might be a useful model for research on fertility.

The method is also cheaper and faster than traditional methods using ES cells. “It enables us to use knockout mice as a screening method in a small lab such as ours,” said Takada. His group is planning to use TALEN technology in the future to identify master regulatory genes based on candidates they found in an earlier expression study.

References

(1) Sung, Y. H.et al. Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol 31, 23–24 (2013)),

(2) Kato T, Miyata K, Sonobe M, Yamashita S, Tamano M, Miura K, Kanai Y, Miyamoto S, Sakuma T, Yamamoto T, Inui M, Kikusui T, Asahara H, Takada S. Production of Sry knockout mouse using TALEN via oocyte injection. Sci Rep. 2013 Nov 5;3:3136