B6.Cg-Sftpa1^tm2Haw Sftpd^tm2Haw/J

Stock number: 027304
Product name: SP-AD KO
Research areas: Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools

Description

These SPAD knock-out mice exhibit progressive lung inflammation and impaired pulmonary clearance of bacteria. They are suitable for use in applications related to lung surfactant homeostasis, innate immunity and pulmonary inflammatory response.

Detailed description

The Sftpd gene encodes the C-type lectin glycoprotein surfactant protein D, which is secreted by pulmonary alveolar type II cells and nonciliated airway cells. It coats bacteria and viruses to promote phagocytosis. The Sftpa1 gene encodes surfactant associated protein A1, which is similar to surfactant protein D, and has a role in innate immune system and provides negative feedback to limit secretion of pulmonary surfactant. These SPAD KO mice carry knock-out alleles for the Sftpd and Sftpa1 genes. ES cells carrying a knock-out allele of Sftpa1, in which exons 2, 3, and 4 and flanking intronic sequence were replaced by a PGK-NEO cassette, were re-targeted with a construct that replaced exon 2 of the Sftpd gene with a PGK-NEO cassette. The double knock-out mice exhibit increased lung inflammation with progressive increase in bronchoalveolar lavage phospholipid, protein, and macrophage and monocyte numbers. At 3 weeks of age, SPAD KO mice exhibit patchy lung inflammation. By 16 weeks of age, interalveolar septum thickening is observed. Double homozygotes exhibit delayed pulmonary clearance of bacterial infection (Pseudomonas aeruginosa). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.

Development

A targeting vector containing a PGK-NEO cassette was used to disrupt exons 2, 3, and 4, including the translation start site and short segments of flanking intronic sequence of the Sftpa1 gene. The construct was electroporated into C57BL/6J x (Rb(11.16)2H x Rb(16.17)32Lub)F1 derived CB1-4 embryonic stem (ES) cells. Correctly targeted ES cells that were heterozygous for the Sftpa1 knock-out allele were then retargeted and electroporated with a construct containing a PGK-NEO cassette to disrupt exon 2, along with the translation start site, of the Sftpd gene. Correctly targeted ES cells containing both knock-out alleles were injected into CD-1 blastocysts. The resulting chimeric male animals were crossed to albino CD-1 female mice, and then backcrossed to C57BL/6 for an unknown number of generations by the donating lab (see SNP note below). Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

Allele

Symbol: Sftpa1^tm2Haw
Name: targeted mutation 2, Samuel Hawgood
MGI accession ID: MGI:2677968
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Sftpa1
Marker name: surfactant associated protein A1
Chromosome: 14
Strain of origin: C57BL/6J x (Rb(11.16)2H x Rb(16.17)32Lub)F1
Molecular note: Exons 2, 3, and 4, including the translation start site and short segments of flanking intronic sequence were replaced with a PGK-neo cassette. Correctly targeted ES cells were subsequently used to target Sftpd thus producing double mutant ES cells.

Allele

Symbol: Sftpd^tm2Haw
Name: targeted mutation 2, Samuel Hawgood
MGI accession ID: MGI:2677969
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Sftpd
Marker name: surfactant associated protein D
Chromosome: 14
Strain of origin: C57BL/6J x (Rb(11.16)2H x Rb(16.17)32Lub)F1
Molecular note: Using ES cells in which a correctly targeted modification of the Sftpa gene had already been created, exon 2 along with the translation start site, was replaced with a PGK-neomycin cassette. Northern blot analysis demonstrated the absence of any Sftpa mRNA in any doubly homozygous mice as well as the absence of full length Sftpd mRNA. A small amount of truncated mRNA for Sftpd lacking the translation start site was seen. Western blot analysis confirmed that no protein products were produced.
General note: This allele was generated in ES cells containing Sftpa1^tm2Haw.