Targeted deletion of miR-182, an abundant retinal microRNA.
OBJECTIVE
MicroRNA-182 (miR-182) is expressed abundantly in the mammalian retina and is therefore thought to perform important roles for the retinal development and the function. To test this hypothesis, we generated miR-182 knockout mice.


METHODS
northern blotting was performed to confirm the robust expression of miR-182 in the eye. The precursor sequence of miR-182 was replaced by the neomycin resistance gene under the control of the phosphoglycerate kinase 1 promoter in a targeting construct. The targeting vector was linearized and transfected into embryonic stem (ES) cells. Recombinant ES clones were selected and injected into blastocysts to generate male chimeras. Heterozygous and homozygous mice were obtained after five generations of backcrossing and were confirmed using genotyping and northern blotting.


RESULTS
Heterozygous (+/-) and homozygous (-/-) knockout mice were morphologically normal, viable, and fertile. Immunohistochemical analysis of the miR-182-deficient retinas did not reveal any apparent structural abnormalities in the retinas. Consistently, global expression profiling using a repeated microarray did not identify significant fluctuations for potential target genes.


CONCLUSIONS
We successfully generated miR-182 knockout mice and characterized the resulting miR-182-depleted retina. This is the first report describing the targeted deletion of a single miRNA that is highly expressed in the retina. The absence of significant transcriptional and phenotypic changes in miR-182-depleted retinas suggests that miR-182 is not a major determinant of retinal development or delamination. Further studies are required to elucidate any functional changes in the retina.
Jin
Zi-Bing
ZB
Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
Hirokawa
Go
G
Gui
Le
L
Takahashi
Rie
R
Osakada
Fumitaka
F
Hiura
Yumiko
Y
Takahashi
Masayo
M
Yasuhara
Osamu
O
Iwai
Naoharu
N
D000818:Animals; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000502:physiology*; D012160:Retina / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*
D035683:MicroRNAs; C540524:Mirn182 microRNA, mouse