In this study, we identified the genomic insertion site of the α-Cre transgene on mouse chromosome 7 by TLA analysis. Inspired by these studies, especially the work identifying the insertion sites for three transgenes, which label some retinal cells 18 and for seven Cre transgenes 19, we decided to use TLA to search the insertion site of the α-Cre transgene. TLA can generate complex DNA libraries covering >100 kb of contiguous sequence surrounding one primer pair complementary to a short locus-specific sequence 24. TLA selectively amplifies and sequences entire genes based on the crosslinking of physically proximal sequences and works without detailed prior locus information 23. Recently, several studies have employed targeted locus amplification (TLA) to map integration sites of different Cre or other transgenes 18, 19, 20, 21, 22. However, these methods are inconvenient 18. This result prompted us to investigate the precise location of the α-Cre transgenic insertion site and any possible functional consequences of the genetic recombination.Ĭommonly used methods for identifying integration sites include inverse PCR 13, 14, 15, whole-genome sequencing and capture-based targeted re-sequencing 16, 17. Of 96 pups, we obtained 50 pups with α-Cre and 48 pups with Bax −/− alleles as predicted, but only five pups (5.2%) with α-Cre Bax −/−, far less than the expected 24 pups (25%), suggesting a genetic linkage between the α-Cre transgene and the Bax locus on chromosome seven 7. To generate α-Cre Rb f/f Bax −/− mice, we first generated α-Cre Rb f/f Bax +/− males and Rb f/f Bax −/− females, and inter-bred them. Bax is a member of Bcl2 family proteins and mediates neuronal cell death 8, including physiological retinal apoptosis 9, 10 and neuronal death in the Rb/p107 double knockout brain 11. In order to elucidate the mechanism of RbKO-induced retinal cell death, we bred α-Cre Rb f/f mice with Bax −/− mice 7. Previously, we reported that Rb gene knockout ( KO) in the retina with α-Cre ( α-Cre Rb f/f) induces ectopic division and apoptosis of many retinal excitatory neurons 6. Such insight is helpful to deduce whether the transgene might have deleterious effects, and to define primer pairs around the integration site to determine whether breeders are heterozygous or homozygous for the transgene. Therefore, the integration site of the α-Cre transgene is random and unknown. The α-Cre mouse line is generated via pronuclear microinjection of the α-Cre transgene construct 3. In the mature eye, the α-Cre is also active in cells of the ciliary body 3, 4 and GABAergic amacrine cells 5. The α-Cre transgene is active from embryonic day 10 in peripheral retinal progenitors that give rise to all cells in the mature retina of this region 3, 4. In addition to the α-enhancer, the α-Cre transgene construct also has the P0 promoter of the Pa圆 gene, an IRES-GFP cassette, an intron from the Hbb (β-hemoglobin) gene, and an SV40 poly (A) sequence 3. The Tg(Pa圆-cre,GFP) 2P gr ( α-Cre) mouse (MGI:3052661) is a commonly used retina-specific Cre line 3. Many retinal cell-targeted Cre transgenic mice have been created to facilitate the study of retinal development and retinal diseases 2. Both spatial and temporal genetic manipulations can be performed using cell type-specific enhancers/promoters to drive Cre recombinase expression in combination with Cre recognition (loxP) sites in interest genes 1. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.Ĭre/LoxP technology is widely used in the field of mouse genetics. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. RT-PCR and buried food pellet test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Most α-Cre + olfactory neurons do not express Pa圆, implicating the influence of neighboring regulatory elements. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in Gα 0 + olfactory neurons. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Further analyses revealed four tandem copies of the transgene. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. The Tg(Pa圆-cre,GFP) 2Pgr ( α-Cre) mouse is a commonly used Cre line thought to be retinal-specific.
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