Neuronal untranslated BC1 RNA: targeted gene elimination in mice

Abstract

Despite the potentially important roles of untranslated RNAs in cellular form or function, genes encoding such RNAs have until now received surprisingly little attention. One such gene encodes BC1 RNA, a small non-mRNA that is delivered to dendritic microdomains in neurons. We have now eliminated the BC1 RNA gene in mice. Three independent founder lines were established from separate embryonic stem cells. The mutant mice appeared to be healthy and showed no anatomical or neurological abnormalities. The gross brain morphology was unaltered in such mice, as were the subcellular distributions of two prototypical dendritic mRNAs (encoding MAP2 and CaMKIIalpha). Due to the relatively recent evolutionary origin of the gene, we expected molecular and behavioral consequences to be subtle. Behavioral analyses, to be reported separately, indicate that the lack of BC1 RNA appears to reduce exploratory activity.

FIG. 1.

Map of the replacement targeting vector and Southern blot of mouse tail DNA. (A) Wild-type genomic locus, genomic clone B122, targeting vector pBC1/Neo-TK, and modified genomic locus lacking the BC1 RNA gene. Neo, neomycin phosphotransferase gene; HSV-TK, TK gene under control of the simian virus 40 promoter. The bars indicate probes for Southern blot hybridization: HR, homologous recombination probe; Neo, probe specific for the neomycin phosphotransferase gene. B, BglII; E, EcoRI; V, EcoRV; H, HindIII; P, PstI. (B) Southern blot analysis of PstI-digested genomic DNAs from nine different pups after crossing of two heterozygous parents. Mice 4 and 9 possess both wild-type alleles (12 kb); mice 1, 2, 6, and 8 are heterozygous; and mice 3, 5, and 7 have both gene deletion alleles (4.8 kb).

FIG. 1.

Map of the replacement targeting vector and Southern blot of mouse tail DNA. (A) Wild-type genomic locus, genomic clone B122, targeting vector pBC1/Neo-TK, and modified genomic locus lacking the BC1 RNA gene. Neo, neomycin phosphotransferase gene; HSV-TK, TK gene under control of the simian virus 40 promoter. The bars indicate probes for Southern blot hybridization: HR, homologous recombination probe; Neo, probe specific for the neomycin phosphotransferase gene. B, BglII; E, EcoRI; V, EcoRV; H, HindIII; P, PstI. (B) Southern blot analysis of PstI-digested genomic DNAs from nine different pups after crossing of two heterozygous parents. Mice 4 and 9 possess both wild-type alleles (12 kb); mice 1, 2, 6, and 8 are heterozygous; and mice 3, 5, and 7 have both gene deletion alleles (4.8 kb).

FIG. 2.

Coronal sections of brains from BC1-deficient and normal mice show no apparent morphological differences. (A) Hippocampal CA1 to -3 fields and dentate gyrus (DG) are indicated. (B) Major brain structures are identified. (C) Amygdala. LaDL, lateral amygdaloid nucleus, dorsolateral; LaVM, lateral amygdaloid nucleus, ventromedial; LaVL, lateral amygdaloid nucleus, ventrolateral; BLP, basolateral amygdaloid nucleus, posterior; BMP, basomedial amygdaloid nucleus, posterior; BLV, basolateral amygdaloid nucleus, ventral. All abbreviations are according to Franklin and Paxinos (15).

FIG. 3.

Northern blot analysis of RNAs extracted from different tissues of BC1-deficient mice. (A) Hybridization with a probe complementary to the 5′ ID domain of BC1 RNA (SB358). Lanes B, T, and L, RNA samples extracted from brain, testes, and liver, respectively, of homozygous BC1-deficient mouse lines 6 and 13. Lane CB, RNA extracted from brains of control mice which exhibits BC1 RNA of ∼150 nt. The size marker positions are shown in nucleotides on the right. (B) After exposure on a phosphorimager, additional hybridization signals corresponding to RNAs migrating faster than BC1 RNA are observed. Arrowheads identify signals at ∼110, ∼90, and ∼75 nt. (C) Hybridization with a probe complementary to the 3′ unique domain of BC1 RNA (probe 90).

FIG. 4.

In situ hybridization of frontal brain sections from BC1-deficient and normal mice with CaMKIIα and MAP2 mRNA probes. CaMKIIα and MAP2 mRNAs show a normal dendritic distribution in BC1-deficient mice. (A to D) Hybridization with a CaMKIIα antisense probe; (E to H) hybridization with a MAP2 antisense probe. All sections are counterstained with cresyl violet. (B and F) Sections from BC1-deficient mice (line 15); (C and G) sections from line 6. (A, E, D, and H) Sections from control mice. Dark-field (B, C, D, F, G, and H) and bright-field (A and E) micrographs are shown. C1, field CA1 of hippocampus; Mol, molecular layer dentate gyrus; PoDG, polymorph layer dentate gyrus; DG, dentate gyrus; GrDG, granular layer dentate gyrus. All abbreviations are according to Franklin and Paxinos (15).