Retinal degeneration mutation in Sftpa1tm1Kor/J and Sftpd -/- targeted mice

Abstract

Surfactant proteins are important collectin immune molecules with a wide distribution throughout the body, including the ocular system. Mice with gene deletions for the surfactant protein genes Sftpa1 and Sftpd were observed to have visual impairment and thinning of the outer nuclear layers of the retina. We hypothesized that gene deletion of Sftpa1 and Sftpd (Sftpa1tm1Kor/J and Sftpd-/-) results in early retinal degeneration in these mice. Sftpa1tm1Kor/J and Sftpd-/- retinas were evaluated by histopathology and optical coherence tomography (OCT). Retinas from Sftpa1tm1Kor/J and Sftpd -/- mice showed early retinal degeneration with loss of the outer nuclear layer. After screening of mice for known retinal degeneration mutations, the mice were found to carry a previously unrecognized Pde6brd1 genotype which resulted from earlier breeding of the strain with Black Swiss mice during their generation. The mutation was outbred and the genotype of Sftpa1tm1Kor/J and Sftpd-/- was confirmed. Outbreeding of the Pde6brd1 mutation resulted in restoration of normal retinal architecture confirmed by in vivo and in vitro examination. We can therefore conclude that loss of Sftpa1 and Sftpd do not result in retinal degeneration. We have now generated retinal Sftpa1 and Sftpd targeted mice that exhibit normal retinal histology.

Fig 1. H&E evaluation and comparison of histological morphology of WT C57Bl/6J and Sftpa1^tm1Kor/J rd1^-/- mouse retinas at 3 months of age.

The retina from Sftpa1^tm1Kor/J rd1^-/- has attenuation of cellular layers and apparent thickening of the RPE. There also appears to be thickening of the optic nerve layer overlying the ganglion cell bodies. (GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, IS inner segments, OS outer segments, RPE retina pigmented epithelium, Chor choroid). Image captured at 40X.

Fig 2. Comparison of retina morphology via SD-OCT (A) and bar graph (B) of 6-week-old WT C57Bl/6J mouse retina and Sftpa1^tm1Kor/J rd1^-/- and the outbreed Sftpa1^tm1Kor/J rd1^+/+.

The significant attenuation of retinal thickness as well as obliteration of nuclear layers in the retinas from Sftpa1^tm1Kor/J rd1^-/- corresponds to a decrease of total retinal thickness by approximately 50%. * p-value is 0.0001 (N = 3).

Fig 3. Confirmation of genotype for the Sftpa1, Sftpd and Mbl1 gene.

(A) Schematic representation of the SP-A encoding gene in wild type (C57BL/6J, upper) and Sftpa1^tm1Kor/J mice (lower). Exons I-VI with encoded Start and Stop codons of the derived mRNA, primer binding sites and expected fragment sizes for genotyping PCR are shown. Agarose gel with correspondent PCR fragments confirms the two genotypes. DNA from Wild type DNA and Sftpa1^tm1Kor/J rd1^+/+ as template were combined with the primer pairs 18889/18890 (lanes 1 and 4), 18889/12834 (lanes 2 and 5) and SPAex6For/SPAex6Rev (lanes 3 and 6), respectively. (B) Wild type gene (upper) and knock out of the Sftpd gene (lower). Wild type DNA and Sftpd^-/- rd1^+/+ DNA as template were combined with the primer pairs SpdFOR/SpdREV (lanes 1 and 4), SpdFOR2/SpdNEOREV2 (lanes 2 and 5) and SPDex8For/SPDex8Rev (lanes 3 and 6), respectively. (C) Confirmation of the integrity of mannose binding lectin gene in wild type, Sftpa1^tm1Kor/J and Sftpd^-/- mice. Primer binding sites for Mbl1 in the gene cluster with the flanking Sftpa1 and Sftpd genes are shown. DNA of wild type, Sftpa1^tm1Kor/J and Sftpd^-/- were combined with the primer pairs Mbl1F1/Mbl1R1 (lanes 1, 3 and 5) and Mbl1F2/Mbl1R2 (lanes 2, 4 and 6), respectively. Primer sequences are found in the S1 Table.

Fig 4. Representative agarose gels with fragments showing genotyping results during outbreeding of the Pde6b^rd1 mutation in Sftpa1^tm1Kor/J and Sftpd ^-/- mice.

(A) Retinal degeneration Pde6b^rd1 with a DdeI digest after PCR resulting in specific fragment sizes for the mutated gene. Lane 1: Pde6brd1^+/+ (298 bp), lane 2: Pde6b^rd1 (104, and137 bp). (B) Retinal degeneration rd8 genotype: DNA templates with primer pairs Crb1-mF1/Crb-mR for rd8^+/+ (lanes 1 and 2) and Crb1-mF2/Crb-mR for rd8^-/- (lanes 3 and 4). (C) Different RPE65 genotypes: (1) methionine/leucin (2) homozygous methionine (3) homozygous leucine.

Fig 5. H&E evaluation and comparison of histological morphology of WT C57BL/6J and Sftpa1^tm1Kor/J rd1^+/+ mouse retinas at 3 months of age.

The outbreeding of Pde6b^rd1 mutation successfully restores the WT histological phenotype to the mouse retina. (GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, IS inner segments, OS outer segments, RPE retina pigmented epithelium, Chor choroid). Image captured at 40X.