Michelle N. Rheault*,
Stefan M. Kren,
Beth K. Thielen,
Hector A. Mesa,
John T. Crosson,
William Thomas,
Yoshikazu Sado||,
Clifford E. Kashtan* and
Yoav Segal
*Division of Pediatric Nephrology, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota; Division of Renal Diseases and Hypertension, Department of Medicine, University of Minnesota, Minneapolis, Minnesota; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota; Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, Minnesota; and ||Shigei Medical Research Institute, Okayama, Japan
Correspondence to Dr. Yoav Segal, MMC 736, 420 Delaware Street SE, Minneapolis, MN 55455. Phone: 612-626-6654; Fax: 612-626-3840; E-mail: ysegal{at}tc.umn.edu
ABSTRACT. X-linked Alport syndrome (XLAS) is a progressive disorderof basement membranes caused by mutations in the COL4A5 gene,encoding the 5 chain of type IV collagen. A mouse model of thisdisorder was generated by targeting a human nonsense mutation,G5X, to the mouse Col4a5 gene. As predicted for a nonsense mutation,hemizygous mutant male mice are null and heterozygous carrierfemale mice are mosaic for 5(IV) chain expression. Mutant malemice and carrier female mice are viable through reproductiveage and fertile. Mutant male mice died spontaneously at 6 to34 wk of age, and carrier female mice died at 8 to 45 wk ofage, manifesting proteinuria, azotemia, and progressive andmanifold histologic abnormalities of the kidney glomerulus andtubulointerstitium. Ultrastructural abnormalities of the glomerularbasement membrane, including lamellation and splitting, werecharacteristic of human XLAS. The mouse model described hererecapitulates essential clinical and pathologic findings ofhuman XLAS. With 5(IV) expression reflecting X-inactivationpatterns, it will be especially useful in studying determinantsof disease variability in the carrier state.
Alport syndrome (AS) is a progressive, hereditary disorder ofbasement membranes for which there is currently no effectivetherapy. Classic features include deafness, anterior lenticonus,and progressive glomerulopathy resulting in end-stage kidneydisease (ESKD). AS results from abnormalities in type IV collagen,a major constituent of basement membranes.
Six genes, COL4A1 to COL4A6, encode six chains of type IV collagen,1(IV) to 6(IV). The genes are arranged in three pairs, COL4A1-COL4A2,COL4A3-COL4A4, and COL4A5-COL4A6, situated, in humans, on chromosomes13, 2, and X, respectively (1). Each gene pair is oriented headto head, with intervening promoter and transcriptional regulatorysites (2,3). The (IV) chains share structural features, includinga noncollagenous amino-terminal sequence of 20 amino acids (7S),a collagenous domain of 1400 amino acids containing multipleGly-X-Y repeats, and a noncollagenous carboxy-terminal (NC1)domain of 230 amino acids. Type IV chains self-assemble to formtriple helices. Three heterotrimers that occur in mammalianbasement membranes have been described: 1-1-2, 3-4-5, and 5-5-6(4,5). The heterotrimers form complex three-dimensional networksthat, with laminin, entactin/nidogen, and proteoglycans, constitutebasement membrane suprastructures (6). The 1-1-2 network ispredominant in the developing glomerular basement membrane untilthe capillary loop stage, when it is substantially replacedby a 3-4-5 network (7,8). Absence or disruption of the 3-4-5network is the underlying abnormality in AS.
X-linked AS (XLAS), caused by mutations in COL4A5, comprises80% of all known cases (9). All affected men progress to ESKD.Although female carriers tend to have a more benign course,12% develop ESKD by age 40, and the risk of ESKD by age 60 approaches30% (10). Various mutations of the COL4A5 gene have been described,including rearrangements, missense mutations, and nonsense mutations(11). Mutations of the 5(IV) chain in men leads to absence ordisruption of the entire 3-4-5 network via mechanisms that areincompletely understood. In female carriers, a mosaic patternof 5(IV) expression is present in glomerular and epidermal basementmembranes, reflecting X-inactivation. Accounting for the balanceof cases, autosomal AS is caused by mutations in COL4A3 or COL4A4.Mutations in these genes lead to loss or disruption of the 3-4-5network, similar to XLAS.
Lack of a mouse model of XLAS has been a barrier to better understandingthe pathophysiologic changes that occur in affected males andfemales as a consequence of primary 5(IV) loss. This articledescribes the first mouse model of XLAS.
Production of Col4a5 Knock-in Mice
We targeted a known human mutation to the murine Col4a5 gene,situated on the X chromosome. An exon 1 transversion, 213GT,predicted to convert codon 5 from glycine to a stop codon (G5X),has been described in a 15-yr-old boy who exhibited full renal,ocular, and auditory manifestations of AS (12). The targetingconstruct pG5X is shown schematically in Figure 1. Short andlong arms of homology were amplified by PCR from 129/SvJ mousegenomic DNA on the basis of available sequence (GenBank accessionNT_085818.1), and cloned, with loxP sites, adjacent to a neomycin-resistancecassette (neoR). The point mutation was introduced by site-directedmutagenesis into codon 5, conserved in mouse Col4a5.
Figure 1. Targeting strategy and genotyping for the Col4a5 mutation. (A) Predicted alleles following homologous recombination in embryonic stem cells and Cre recombination in vivo are shown. Exons, neoR, and PCR genotyping primers (arrows) are indicated. In the Southern hybridization strategy, an external probe identifies the modified locus as an aberrant AflII (A) restriction fragment. (B) Allele-specific PCR. Amplicons of differing size are produced by allele-specific primers complementary at their 3'-end bases to the wild-type (ASPwt; C) or mutant (ASPG5X; T) base. Additional mismatches, determined experimentally for optimal single-tube reaction conditions, are in bold. *G5X mutation. (C) Genotyping from representative litters by Southern blotting and allele-specific PCR assays, using the primer set F1/ASPwt/ASPG5X/R1, or F2/R2. Characteristic patterns are shown for wild-type animals (Y/+, +/+), mutant male mice (Y/–), and female carrier mice (+/–).
The targeting construct was introduced via electroporation intomouse embryonic stem cells, and stable clones were selectedin G418-containing media. Four positive clones were identifiedby PCR and Southern blotting, among which two harbored the pointmutation. One clone, injected into C57BL/6 blastocysts, produced14 high-coat color chimeras, 11 male and 3 female, that reachedreproductive age. Three male chimeras transmitted the mutantallele to female offspring. Female carriers selected from amongthese first-generation offspring were mated with males fromthe FVB/N-TgN(ACTB-cre)2Mrt strain (The Jackson Laboratories,Bar Harbor, ME), a "deletor" strain expressing Cre recombinaseunder the human -actin promoter (13), thus excising neoR. Germlinetransmission of the neoR-negative allele to succeeding generationswas confirmed by genotyping. Both neoR-positive and -negativelines have been generated by successive backcrosses onto theC57BL/6 background. Animal protocols were approved by the InstitutionalAnimal Care and Use Committee of the University of Minnesota.
Genotyping
Mouse embryonic stem cell and tail DNA was extracted using theEasy DNA Kit (Invitrogen, San Diego, CA). For Southern blotting,5-µg DNA samples were digested, separated on 0.8% agarosegels, and transferred to nylon membranes. Probes external tothe arms of homology were prepared by 32P-dCTP random labeling,and hybridization and subsequent washings were carried out bystandard techniques. For PCR genotyping, we developed a single-tube,allele-specific assay, optimized for PCR conditions, and directedagainst the point mutation. In this assay, a set of four primersyields a 172-bp amplicon from the wild-type allele and a 248-bpamplicon from the mutant allele (Figure 1). A second PCR genotypingassay was developed to detect the single remaining loxP sitein mice from the neoR-negative line (Figure 1). For this assay,primers were as follows: F2, 5'-TTTGTTTGTAGGTCCTTTCATACATC-3';and R2, 5'-GTGTATTTATTGATTGTATTGTGACTTGG-3'. Cycling conditionsconsisted of denaturation at 94°C for 30 s, followed by28 cycles of denaturation at 94°C for 30 s, and combinedannealing and polymerization at 68°C for 20 s. Productswere resolved on 3.5% agarose gels.
Laboratory Analysis
Mice were anesthetized by intraperitoneal injection of pentobarbital(50 mg/kg). Blood and urine samples were obtained by punctureof the vena cava and bladder, respectively. Urine protein wasmeasured using the Bio-Rad Protein Assay (Richmond, CA). Urinecreatinine was measured by the Jaffé reaction, usingthe Creatinine Analyzer-2 (Beckman Coulter). Plasma urea nitrogenwas measured using the Liquid Urea Nitrogen Reagent Set (PointeScientific).
Histology
Animals were anesthetized, and kidneys were perfused via theabdominal aorta with 1.25% glutaraldehyde solution before removal.For light microscopy, kidneys were embedded in paraffin, cutinto sections, and stained with periodic acid-Schiff reagent,using standard techniques. For electron microscopy, 2- to 3-mmpieces of cortex fixed in 1.25% buffered glutaraldehyde solutionwere processed successively using the Automated Tissue Processor(EMS LYNX) and embedded in a premade Epon 812 resin mixture.Semithin sections were examined to identify glomeruli. Ultrathinsections on copper mesh grids were stained with uranyl acetateand lead citrate using the EM STAIN automatic grid stainer (Leica,Wetzlar, Germany) and examined with a CX-100 transmission electronmicroscope (JEOL, Tokyo, Japan).
Immunofluorescence
Kidneys were removed, immersed in OCT, and snap-frozen in liquidnitrogen vapor. Five-micrometer cryostat sections were placedon slides and postfixed in acetone. Tissues were stained asdescribed previously (14). Epitope-defined rat monoclonal antibodiesdirected against the 1(IV) (H11), 2(IV) (H22), 3(IV) (H31),5(IV) (H53), and 6(IV) (B66) chains were used for immunostaining(15). Secondary FITC-conjugated donkey anti-rat IgG antibodywas obtained from Jackson Immunoresearch (West Grove, PA). Slideswere viewed by fluorescence microscopy.
Ribonuclease (RNase) Protection Assays
Riboprobe templates were amplified by PCR from a mouse kidneycDNA library (Clontech, Palo Alto, CA), and cloned into pBCKS- (Stratagene, La Jolla, CA). With corresponding GenBank accessions,nucleotide (nt) positions, and sizes, these were directed againstmessages for the 1(IV) (XM_134042, nt 3918 to 4311, 394 bases),3(IV) (NM_007734, nt 5554 to 5850, 297 bases), 4(IV) (NM_129902,nt 4832 to 5050, 219 bases), and 5(IV) (XM_136081, nt 2101 to2257, 157 bases) chains and L32 (NM_172086, nt 369 to 483, 115bases). Templates were verified by direct sequencing. Riboprobeswere synthesized by transcription of linearized template mixtureswith T7 RNA polymerase (Ambion, Austin, TX). Total RNA was extractedfrom whole mouse kidney using Ultraspec RNA (Biotecx Laboratories).Twenty-microgram samples were subjected to assay using the RPAIIIKit (Ambion). Protected fragments were resolved by electrophoresisin denaturing 6% polyacrylamide gels. Gels were scanned by phosphorimaging(Molecular Dynamics, Sunnyvale, CA), and bands were quantifiedusing ImageQuant (Molecular Diagnostics). Band densities wereadjusted for the length of the probe and expressed relativeto L32.
Statistical Analyses
Genotype distributions were compared with expectations fromMendelian ratios by 2 analysis. Survival curves for mutant malemice and female carrier mice were estimated by Kaplan-Meierproduct-limit estimates, censoring mice that were killed atpredetermined time points. Curves were compared by the log-ranktest. Message levels measured by RNase protection assay werecompared by t test.
Pedigrees
On the basis of a human mutation (12), we introduced the G5Xnonsense mutation into the signal peptide sequence of the mouseCol4a5 gene. Results are presented for Col4a5-targeted mouselines through the ninth generation of backcrossing onto theC57BL/6 background. Mutant male mice and female carrier micewere viable and fertile, with the ability to transmit the mutationto offspring. Genotyping assays for representative litters areshown in Figure 1. Examination of the resultant pedigrees forthe neoR-positive line and the neoR-negative line, generatedby Cre recombinase-mediated excision of the neoR cassette, showedthe expected Mendelian segregation ratio of 1:1:1:1 (Table 1).
Table 1. Distribution of genotypes for Col45-targeted mouse lines
By immunofluorescence, we detected 1(IV), 2(IV), 3(IV), and5(IV) chain expression within glomerular and tubular basementmembranes of wild-type kidneys (Figure 2), consistent with previouslydescribed patterns (16). Expression of the 5(IV) chain was lostin kidneys from mutant male mice, confirming predicted effectsof the targeted mutation. Basement membrane expression of the3(IV) chain was also lost, whereas that of the 1(IV) and 2(IV)chains was conserved. Expression of the 6(IV) chain, detectedwithin Bowman’s capsule in wild-type mice, was lost (notshown). Expression of the 5(IV) and 3(IV) chains in kidneysfrom female carriers was mosaic, reflecting X-inactivation.Effects of the targeted mutation on type IV collagen expressionwere similar in the neoR-positive and -negative lines and wereconsistent with those of spontaneously occurring XLAS mutationsin humans and dogs (17).
Figure 2. Immunostaining for type IV collagen chains in kidneys of wild-type male mice (A, D, G, and J), mutant male mice (B, E, H, and K), and female carrier mice (C, F, I, and L). Expression of the 1(IV) (A, B, C) and 2(IV) (D, E, F) chains was conserved in mutant male mice and female carrier mice. Expression of the 3(IV) (G, H, I) and 5(IV) (J, K, L) chains was present in wild-type male mice, absent in mutant male mice, and present in a mosaic pattern in female carrier mice.
Clinical Course of Col4a5-Targeted Mice
There were no obvious differences in clinical or pathologiccourse between the neoR-positive and -negative lines; therefore,the lines were combined for further analysis.
All animals seemed normal at birth through weaning and reproductiveage. Hemizygous mutant male mice demonstrated signs of illnessand died spontaneously at 6 to 34 wk of age, without survivorsthereafter. Heterozygous female carriers died at 8 to 45 wkof age, with survivors alive out to 50 wk. Comparison of estimatedsurvival curves for mutant male mice and female carrier micewere significantly different (2 = 43.1, P < 0.001), divergingat 15 wk of age (Figure 3). Estimated median survivals for mutantmale mice and female carrier mice were 23 and 39 wk, respectively.
Figure 3. Estimated survival functions for mutant male mice and female carrier mice.
Mutant male mice and female carrier mice developed progressivefunctional and histologic abnormalities of the kidneys, likelythe major cause of death. Proteinuria, defined arbitrarily asurine protein excretion >10 mg/mg creatinine, was rarelyobserved in wild-type mice. By contrast, 97% of mutant malemice that were examined after 7 wk of age (n = 63) and 78% offemale carrier mice that were examined after 9 wk of age (n= 50) had proteinuria (Figure 4). Plasma urea nitrogen measurementsalso segregated mutant male mice and female carrier mice fromwild-type littermates, by inspection (Figure 5).
Figure 4. Urine protein excretion in mutant male mice with wild-type littermate controls (A) and female carrier mice with wild-type littermate controls (B).
Figure 5. Plasma urea nitrogen in mutant male mice with wild-type littermate controls (A) and female carrier mice with wild-type littermate controls (B).
Pathologic Findings in Col4a5-Targeted Mice
Kidneys from mutant male mice and female carrier mice were grosslyabnormal at end stage, exhibiting surface pallor, dullness,and pockmarking (not shown). By light microscopy, we observedprogressive pathologic changes (Figure 6). Kidneys from mutantmale mice at 4 wk of age displayed only subtle pathologic findingsof capillary wall thickening and mesangial hypercellularity,with overall sparing of the tubulointerstitium. By 17 wk ofage, widespread interstitial inflammation with tubular atrophyand dilation was present. At this stage, glomeruli exhibitedvariable and manifold abnormalities, including capillary loopdilation and simplification, capillary tuft collapse, capsularadhesions, and focal sclerosis. Rarely, crescents were present.Kidneys from female carrier mice also demonstrated progressionof pathologic abnormalities, over an extended time scale, bycomparison. Kidney sections from a female carrier at 17 wk ofage revealed focal abnormalities of the glomeruli and tubulointerstitium(Figure 6).
Figure 6. Histopathologic changes in Col4a5-targeted mice. See text for description. Periodic acid-Schiff-stained kidney sections were prepared from wild-type (A and C) and mutant (B and D) male mice at 4 wk of age, wild-type (E) and mutant (F and G) male mice at 17 wk of age, and wild-type (H) and carrier (I) female mice at 17 wk of age. (I) Regions of focal glomerular sclerosis (long arrow) and interstitial expansion (short arrows) are indicated. Scale bars: 200 µm in A, applies to A, B, E, F, H, and I; 50 µm in C applies to C, D, and G.
Lamellation of glomerular basement membranes, a hallmark ultrastructuralfeature of AS, was evident in kidney sections from mutant malemice as young as 4 wk (Figure 7). By 17 wk of age, diffuse glomerularbasement membrane abnormalities, including lamellation and splitting,were fully evident. Concomitant evidence of podocyte injuryincluded foot process effacement, vesiculation, and denudation.Study of a female carrier at 17 wk of age revealed focal areasof lamellation within some glomeruli and more diffuse changeswithin others (Figure 7). Study of a female carriers at 30 wkof age revealed widespread changes (not shown).
Figure 7. Ultrastructural changes of the glomerular basement membrane. Glomerular capillary loops from wild-type male and female mouse kidneys are depicted in A and D, respectively. In those from mutant male mice at 4 wk of age (B), regions of glomerular basement membrane lamellation are present (arrow). By 17 wk, diffuse splitting and podocyte foot process fusion are evident (C). Female carrier mice demonstrate variability in glomerular basement appearance at 17 wk, with some glomerular capillary loops displaying only focal thickening and splitting (arrow; E), whereas others show widespread changes (F).
Effects of the Col4a5 Mutation on Type IV Collagen mRNA Levels
To begin addressing detailed mechanisms of the nonsense mutation,we measured type IV collagen chain mRNA levels in kidneys fromweanling mice of the neoR-negative line. Levels for 1(IV), 3(IV),and 4(IV) chain mRNA were not significantly different betweenwild-type and mutant male mice (Figure 8). The 5(IV) mRNA level,however, was significantly lower in mutant male mice, suggestingeffects of the mutation on RNA synthesis and/or degradation.
Figure 8. RNase protection assays for collagen IV. A representative gel is shown for the multiprobe RNase protection assay for wild-type (Y/+) and mutant (Y/–) male mice. Results are summarized for the two groups (n = 5 wild-type and 7 mutant male mice). The 5(IV) mRNA level was found to be lower in mutant males than in wild-type controls (P < 0.01).
This is, to our knowledge, the first reported mouse model ofXLAS. The clinical, histologic, and immunofluorescence findingsdescribed in this model closely approximate findings in humanXLAS (9). Affected male mice develop a progressive glomerulopathymanifested clinically by proteinuria and azotemia, leading todeath from kidney failure at a young age. Indeed, their clinicalcourse is comparable to those in previously described modelsof autosomal AS, rendered deficient in the type IV collagenminor chains by mutagenesis of the Col4a3-Col4a4 locus (18–20).Female carriers in our model of XLAS display dominant effectsof the targeted mutation, with significant kidney abnormalitieswithin 1 yr of age.
Several animal models of AS have been described (21). Two spontaneousX-linked canine lines have been characterized: the CanadianSamoyed (22) and Navasota mongrel (23) lines. Affected maledogs develop proteinuria within the first 6 mo of life and progressto renal failure and death between 12 and 18 mo. Female carriersdisplay proteinuria at 2 to 3 mo but progress comparativelyslowly, with only a minority progressing to renal ESKD (24).A spontaneous autosomal recessive inherited nephropathy wasdiscovered in an English Cocker Spaniel line (14). Several transgenicmouse models that follow an autosomal recessive inheritancepattern have been created. Two models were created by knockoutof the Col4a3 gene (18,19), and a third was created by randominsertional mutagenesis of the Col4a3 and Col4a4 genes (20).A spontaneous bull terrier canine model is the only candidateautosomal dominant model of AS (25). These models recapitulateclinical features of AS, with some variability. They have provedvaluable in ongoing efforts to understand disease pathways (26–31)and develop therapies (28,31–34).
As a novel mouse model of the XLAS, our Col4a5-targeted linesshould provide new opportunities for elucidating disease mechanisms.In particular, they afford a new model of the female carrierstate, characterized by mosaic patterns of basement membranetype IV collagen expression. Determinants of disease progressionin human XLAS carriers are largely unknown and likely to differfrom those in male counterparts. Whereas males who share familialmutations tend to progress to ESKD over comparable time frames,female carriers display significantly greater intrafamilialvariability (35). Furthermore, whereas genotype-phenotype correlationscan be drawn in large cohorts of males, no such correlationscan be drawn in comparable studies of female carriers (10).It has been postulated that X-chromosome inactivation patterns,with their influence over type IV collagen expression, figureprominently in determination of the carrier phenotype, but studieshave been few and limited primarily to assessments of extrarenaltissues (36). This mouse model is uniquely suited for studyof this and other clinical questions in the carrier female.It offers the possibilities of assaying kidney-wide X-inactivationpatterns and basement membrane type IV collagen gene expression,as postulated determinants of disease severity, and of skewingX-inactivation experimentally, by breeding strategies, in directedinvestigations.
This report summarizes our findings for Col4a5-targeted linesthrough nine generations of backcrossing onto the C57BL/6 background.The experimental groups are on comparable but mixed geneticbackgrounds. We fully expect that modified clinical and pathologicfeatures of the model will become evident with progressive backcrossingto a congenic background. Indeed, the importance of geneticbackground, as a basis for phenotypic variability, has beenestablished for an autosomal recessive Alport mouse line (26).
Mutant mice in our lines transmit a known human point mutation.This "knock-in" mutation typifies "small" mutations, includingsubstitutions, deletions, and insertions, composing the majorityof XLAS mutations, now >300 in number. As such, the mutantline may offer options for tailoring novel therapies to knowledgeof the specific disease mutation. For the G5X mutation, theseoptions might include nonsense suppression, e.g., using aminoglycosides(37,38), and targeted gene correction, using short gene fragments(39). We have identified effects of the nonsense mutation onmRNA turnover, producing reduced levels of steady-state mRNAin weanling animals. It is likely that tailored therapies willhave to account for this effect, which is lesser in magnitudethan that observed in Samoyed dogs with XLAS as a result ofa premature stop codon (40) but still significant. In contrastto findings in the Samoyed dog model, we did not observe coordinatechanges in 3(IV) and 4(IV) mRNA levels.
In summary, we have described a novel mouse model of XLAS. Thismodel should prove valuable in further elucidating primary basementmembrane abnormalities and their promulgation through cellularelements within the kidney glomerulus and perhaps other tissuestructures as well. The model should have special relevancein determining requirements for the type IV collagen chainsin sustaining kidney function in female carrier mosaics. Finally,the model can serve as a tool in developing therapies, includingbut not limited to tailored counteraction of specific diseasemutations, preservation of glomerular basement membrane performance,and prevention and treatment of secondary tubulointerstitialinjury.
Acknowledgments
This work was supported by National Institutes of Health grantDK60695 and DK07087, a Clinical Scientist Award from the NationalKidney Foundation, and the McKnight Land-Grant Professorship,Grant-in-Aid and Undergraduate Research Opportunities Programsat the University of Minnesota.
Portions of this work were presented at the 2003 meeting ofthe American Society of Nephrology, San Diego, CA.
We thank the members of the Mouse Genetics Laboratory at theUniversity of Minnesota for expert services rendered; Dr. HassanIbrahim for thoughtful guidance and support; and Kristin Loewe,Adrienne Wiebe, and Bing Zhou for experimental assistance.
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Received for publication January 25, 2004.
Accepted for publication March 24, 2004.
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Top
Abstract
Introduction
5 chain of type IV collagen. A mouse model of this disorder was generated by targeting a human nonsense mutation, G5X, to the mouse Col4a5 gene. As predicted for a nonsense mutation, hemizygous mutant male mice are null and heterozygous carrier female mice are mosaic for 
20 amino acids (7S), a collagenous domain of 
T, predicted to convert codon 5 from glycine to a stop codon (G5X), has been described in a 15-yr-old boy who exhibited full renal, ocular, and auditory manifestations of AS (12). The targeting construct pG5X is shown schematically in Figure 1. Short and long arms of homology were amplified by PCR from 129/SvJ mouse genomic DNA on the basis of available sequence (GenBank accession NT_085818.1), and cloned, with loxP sites, adjacent to a neomycin-resistance cassette (neoR). The point mutation was introduced by site-directed mutagenesis into codon 5, conserved in mouse Col4a5. 
-actin promoter (13), thus excising neoR. Germline transmission of the neoR-negative allele to succeeding generations was confirmed by genotyping. Both neoR-positive and -negative lines have been generated by successive backcrosses onto the C57BL/6 background. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. 
2 analysis. Survival curves for mutant male mice and female carrier mice were estimated by Kaplan-Meier product-limit estimates, censoring mice that were killed at predetermined time points. Curves were compared by the log-rank test. Message levels measured by RNase protection assay were compared by t test. 
