Supplementary material (Tanaka et al. 2000)
Fig. 1. LAMP-2 deficient mice: Generation, increased mortality and loss of weight. a.
Strategy for inactivation of the lamp-2 gene by homologous recombination in ES cells. (I)
Partial structure of the genomic locus representing about 12 kbp of the lamp-2 gene region.
The bar represents an external 3´probe. (II) Targeting vector pCK-Lamp-2(neo) with 8 kbp
homology to the lamp-2 gene locus. (III) Predicted lamp-2 gene locus after homologous
recombination. b. Southern blot analysis of ES cell clones. The 3´ probe was hybridized to
HindIII digested genomic DNA from ES cell clones. Since lamp-2 is located on the Xchromosome the lamp-2 wild-type allele (7.5 kb) is lost after homologous recombination. The
8.7 kbp DNA fragment indicates a targeted allele. c. PCR-analysis of tail-genomic DNA with
an exon-specific PCR amplifying a 0.48 kb fragment in LAMP-2+/+, a 0.48kb and a 1.68 kb
fragment in LAMP-2+/- and a 1.68 kb fragment in LAMP-2-/- mice, respectively.
Method: Generation of LAMP-2 deficient mice
A EMBL3 -129SV mouse phage library from Stratagene Inc., La Jolla, USA was screened
with a 390 bp genomic amplification product of mouse lamp-2 consisting of an 372 bp exon
(designated exon ”X”, cDNA position 689 to 1061) flanked by short intronic sequences. The
neo expression cassette from pMC1neopA (Stratagene) was inserted as a XbaI DNA
restriction fragment into a SpeI restriction site located in the exon ”X” of a 8kb genomic
BamHI fragment. The targeting vector was linearized with NotI and introduced into the ES
cell line E14-1 by electroporation. ES-cells were cultured as described 1. G418 resistant
colonies were screened by Southern blot analysis of DNA digested with Hind-III and
hybridized with the 3´ probe (Fig. 1a). Two independent mutated ES lines were microinjected
into blastocysts of C57BL/6J mice. From both cell lines chimeric males with germline
transmission were obtained. Chimeric males were mated to C57BL/6J and 129SV females.
Offspring was genotyped for the lamp-2 gene mutation by Southern blot analysis of Hind III digested genomic DNA, using the 3´ probe or by PCR analyses using a neomycin-specific
PCR2 and an exon-specific-PCR with primers (LII/9 5´agctgaaatgagtaagag 3´ and LII/10 5´
tttgtagcacaccaagca 3´) flanking the exon used for interruption. As controls littermates of the
F2 and F3 generation were used as well as offspring of control mice matched for age and sex.
1. Köster, A. et al. Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in
misrouting of lysosomal proteins. EMBO J. 12, 5219-5223(1993).
2. Saftig, P. et al. Amyloidogenic processing of human amyloid precursor protein in hippocampal neurons
devoid of cathepsin D. J. Biol. Chem. 271, 27241-27244 (1996).
Fig. 2. Accumulation of autophagic vacuoles in LAMP-2 deficient tissues a. Photomicrographs of acinar gland cells of the pancreas in 6.5 month old LAMP-2 deficient mice.
The insert shows a section through a control pancreas. b. Autophagic vacuoles in neutrophilic
polymorphnuclear leukocyte of a 9 month old LAMP-2-/- mouse c. Capillary endothelium of
LAMP-2-/- (21 days old ) kidney containing autophagic vacuoles (arrowheads). d.
Photomicrograph of cardiac muscle of a 19 month old LAMP-2 -/- mouse. Arrows point to
large vacuoles. e. Cardiac muscles of a control and a deficient animal (6.5 month),
respectively. In (f) several condensed mitochondria are seen (arrows), which are contained in
autophagic vacuoles (insert).g. PAS-staining of glycogen (arrows) in a muscle fiber (M.
sternomastoideus) of a 19 month old LAMP-2 -/- mouse h. muscle fiber splitting (arrow) and
degeneration (*) in M. sternomastoideus of a 9 month old LAMP-2 deficient mouse.
Bars: a: 14 m;insert ? m ; b: 0.4 m; c: 0.4 m;d: 22 m;e: 3 m, f:4 m; insert: 1 m; g: 9
m; h: 8 m.