Reciprocal backcross mice confirm major loci linked to hyperoxic acute lung
injury survival time
Daniel R. Prows,1,2Abby V. Winterberg,2William J. Gibbons, Jr.,2Benjamin B. Burzynski,2
Chunyan Liu,3and Todd G. Nick1,3
1Department of Pediatrics, University of Cincinnati College of Medicine, and2Division and Program in Human Genetics
and3Division of Biostatistics and Epidemiology, Children’s Hospital Medical Center, Cincinnati, Ohio
Submitted 5 December 2008; accepted in final form 30 April 2009
Prows DR, Winterberg AV, Gibbons WJ Jr, Burzynski BB,
Liu C, Nick TG. Reciprocal backcross mice confirm major loci
linked to hyperoxic acute lung injury survival time. Physiol
Genomics 38: 158–168, 2009. First published May 5, 2009;
doi:10.1152/physiolgenomics.90392.2008.—Morbidity and mor-
tality associated with acute lung injury (ALI) and acute respiratory
distress syndrome remain substantial. Although many candidate genes
have been tested, a clear understanding of the pathogenesis is lacking,
as is our ability to predict individual outcome. Because ALI is a
complex disease, single gene approaches cannot easily identify effec-
tors that must be treated concurrently. We employed a strategy to help
identify critical genes and gene combinations involved in ALI mor-
tality. Using hyperoxia to induce ALI, a mouse model for genetic
analyses of ALI survival time was identified: C57BL/6J (B) mice are
sensitive (i.e., die early), whereas 129X1/SvJ (S) mice are signifi-
cantly more resistant, but with low penetrance. Segregation analysis
of reciprocal F2 mice generated from B and S strains revealed
significant sex, cross, and parent of origin effects. Quantitative trait
locus (QTL) analysis identified five chromosomal regions signifi-
cantly linked to hyperoxic ALI survival time (named Shali1–Shali5).
Further analyses demonstrated that both parental strains contribute
resistance alleles to their offspring and that the phenotype demon-
strated parent of origin effects. To validate earlier findings, we
generated and tested mice from all eight possible B-S-derived back-
crosses. Results from segregation and QTL analyses of 935 back-
crosses, alone and combined with the previous 840 B-S-derived F2
population, further supported the highly significant QTLs on chromo-
somes 1 (Shali1) and 4 (Shali2) and confirmed that the sex, cross, and
parent of origin all contribute to survival time with hyperoxic ALI.
adult respiratory distress syndrome; mouse model; parent of origin
effects; quantitative trait locus
DESPITE SIGNIFICANT ADVANCES in supportive care and improve-
ments in ventilator management (1), acute lung injury (ALI)
and adult respiratory distress syndrome (ARDS) are still asso-
ciated with 30–45% mortality, which accounts for nearly
75,000 deaths/yr in the United States (36). Although more than
four decades since its first description (2), the persistent high
death rate related to ALI/ARDS continues to be a formidable
challenge. Even results from recent meta-analyses have been
contradictory as to whether ALI/ARDS mortality rates have
changed over the last 15 yr (29, 50). Although anyone at any
age can die after the development and progression of ALI/
ARDS, older patients have an increased risk of dying (36, 39,
40, 51). Accordingly, as the population gets older, the future
social and healthcare burdens surrounding ALI will continue to
escalate. Consequently, alternative research strategies are needed
to address this unremitting problem.
The pathophysiology of ALI has been described in detail
(11, 18, 24, 25, 45, 46), yet little is known about the critical
genes or gene products involved in the pathological mecha-
nisms that lead to death. Because only ?20% of ALI/ARDS
patients die from refractory respiratory failure (15, 26, 43), the
most important factors and mediators resulting in death are not
obvious and may be far removed from lung-related pheno-
types. This could, in part, explain the problems in identifying
factors (and their associated genes) that lead to better prog-
noses. The difficulty is highlighted by the fact that no specific
pharmacological therapies have reduced mortality in large
phase III studies (7). Therefore, with the long-term goal of
significantly decreasing ALI-associated mortality, we em-
barked on a very different strategy to traditional candidate gene
approaches. Rather than predicting and testing genes directly
or indirectly associated with ALI one at a time, quantitative
trait locus (QTL) analysis initially identifies chromosomal
regions of linkage without a priori prediction of the genes
likely to harbor risk variants. Subsequent resolution of these
QTLs will generate new hypotheses-driven studies aimed at
decreasing ALI-associated mortality.
Using continuous hyperoxia, a well-established method to
induce lung injury and death in animals (4, 5, 20, 44, 47), we
(33) recently demonstrated that survival time with hyperoxic
ALI (HALI) was a heritable phenotype in mice. Analysis of
reciprocal F1 mice generated from sensitive C57BL/6J (B)
mice and resistant 129X1/SvJ (S) mice [i.e., B ? S (or B.S)
and S ? B (or S.B)] revealed a parent of origin effect. To
further investigate this parent of origin difference, a large F2
population (n ? 840 mice) was produced, consisting of 197 or
more mice from each of the four possible intercrosses between
B and S inbred mice. QTL analysis of the total F2population
identified five genomic regions (i.e., QTLs) significantly linked
to HALI survival time, designated survival to hyperoxic acute
lung injury 1–5 (Shali1–5) (32). Additional genetic analyses of
F2subpopulations identified significant sex, cross, and parent
of origin effects on HALI survival time and demonstrated that
a further increase in HALI survival time was due to a recom-
bination of resistance alleles originating from both parental
To validate these findings and further examine the mode of
inheritance of HALI survival time, we generated and tested a
large population (n ? 935) of B-S-derived backcross mice.
This population consisted of at least 94 mice from each of the
eight possible backcross breeding schemes (Table 1). Separate
segregation and QTL analyses were performed on males,
females, and all mice for the group S backcrosses (i.e., the four
Address for reprint requests and other correspondence: D. R. Prows, Div. of
Human Genetics, Children’s Hospital Medical Center, 3333 Burnet Ave., Bldg. R,
MLC 7016, Rm. 1464, Cincinnati, OH 45229-3039 (e-mail: daniel.prows
Physiol Genomics 38: 158–168, 2009.
First published May 5, 2009; doi:10.1152/physiolgenomics.90392.2008.
1094-8341/09 $8.00 Copyright © 2009 the American Physiological Society158
backcrosses that yielded SS or SB genotypes), the group B
backcrosses (i.e., the four backcrosses that yielded BB or SB
genotypes), and the total backcross population. Segregation
analyses verified significant cross and sex effects for HALI
survival time among the backcross groups. QTL analyses
results from the total backcross population verified Shali1 and
Shali2 as highly significant linkages. Interestingly, when di-
viding the total population into the group B and S backcrosses,
Shali1 was highly significant in group S backcrosses and
Shali2 was highly significant in group B backcrosses. Overall,
the backcross data validated that the S allele for Shali1 and the
B allele for Shali2 are dominant or additive for HALI resis-
tance. QTL analysis of the combined dataset, consisting of total
B-S-derived backcrosses and F2mice, confirmed Shali1–5 as
MATERIALS AND METHODS
From our initial screen of 18 inbred mouse strains, group B mice
were identified as sensitive, whereas group S mice were significantly
more resistant to HALI-induced mortality, although the penetrance of
the resistance trait was low, ranging from 30% to 35% (33). Crosses
of these strains (the B-S model) were used to generate mice for initial
genetic and segregation analyses and the subsequent QTL analysis.
First, reciprocal F1lines were generated by breeding B females to S
males (B.S) and S females to B males (S.B). The significant pheno-
type difference between these reciprocal F1mice suggested a parent of
origin effect, so reciprocal F1offspring were systematically bred back
to both parental lines, with the goal of generating ?100 mice or more
for each of the eight possible backcrosses (Table 1); a total of 935
backcross mice were used in these genetic experiments. Additional
experiments were performed on a combined dataset consisting of this
backcross population and the previously described (32, 33) F2popu-
lation (n ? 840) in a single group of 1,775 recombinant mice.
Mice in standard shoebox cages with food and water ad libitum
were placed inside a 0.13-m3Plexiglas inhalation chamber (Stellar
Plastics, Detroit, MI) and exposed continuously to ?95% O2 until
death. Each chamber housed up to nine cages with four mice per cage;
individual mice were clearly visible from above and/or the sides. O2
level in the chamber was continuously controlled using a ProOx 110
portable O2monitor (Biospherix, Redfield, NY), which was calibrated
before each exposure using room air and 100% O2. The status of the
exposures was closely monitored, such that the determined survival
time for each mouse was within 5% error. Mice were exposed
between 6 and 12 wk of age, and each exposure was continued until
all mice within the chamber were dead. Mice included in these
experiments were bred and exposed over a period of ?5 yr; no
significant seasonal differences were noted. Mice were handled in
accordance with protocols approved by the Institutional Animal Care
and Use Committee of Cincinnati Children’s Hospital Medical Center.
Genomic DNA was isolated from 0.5-cm tail clips using a com-
mercial DNA extraction kit (Wizard Genomic DNA, Promega, Mad-
ison, WI). A SpectraMAX 190 spectrophotometer (Molecular De-
vices, Sunnyvale, CA) and a 96-well quartz plate were used to analyze
samples for purity (absorbance at 260/280 nm) and DNA content
(absorbance at 260 nm). DNAs were diluted to 20 ng/?l for PCR
requiring agarose gel separation or to 5 ng/?l for PCR using fluores-
Primer pairs for polymorphic markers between the B and S strains
were purchased from Research Genetics/Invitrogen (Frederick, MD)
or IDT (Coralville, IA). PCR was performed in 15-?l volumes in
96-well plates (USA Scientific, Ocala, FL) using a four-block ther-
mocycler (model PTC-225 or PTC-240, Bio-Rad) as previously de-
scribed (33). Markers with PCR product allele sizes of 5% or more
were separated by 2.5–4% agarose (ISC BioExpress, Kaysville, UT)
gels and stained with ethidium bromide. Microsatellite markers ?5%
different in allele size were amplified from 20 ng of total DNA using
fluorescent primers synthesized by ABI and the protocols provided.
Fluorescent PCR products were separated using an ABI-3730xL
sequencer located at the Cincinnati Children’s Hospital DNA Se-
quencing Core Facility (http://dna.chmcc.org/), and genotypes were
ascertained using GeneMapper software (version 3.5, ABI).
Because survival times of the reciprocal F1and F2mice suggested
a parent of origin effect in HALI survival (33), offspring from all eight
possible backcross breeding schemes (Table 1) were generated to
further investigate these cross and sex differences and to help predict
trait heritability. Survival times were assessed in total mice and in
males and females separately for each of the following cohorts: 1) the
total backcross population; 2) the two groups of four combined
backcrosses, based on whether the possible genotypes were heterozy-
gous and homozygous S (group S backcrosses ? SS or SB) or
heterozygous and homozygous B (group B backcrosses ? BB or BS);
3) the four backcross pairs generated with the same inbred strain as
the dam or sire (i.e., S dam ? crosses 9 and 10, S sire ? crosses 3 and
8, B dam ? crosses 4 and 5, and B sire ? crosses 2 and 7); and 4) the
combined backcross plus F2 dataset. Mean survival times (MSTs)
were also assessed for total mice in the eight separate backcrosses.
Cross Comparisons and Assessment of Parental Effects
To confirm the cross differences seen in the F2analysis and further
assess these parent of origin effects, single and paired backcrosses
within group B or S were compared. Within-group comparisons were
valid to assess cross effects, since the genetic compositions of the
lines are similar (i.e., group S backcrosses average 75% S alleles and
25% B alleles and group B backcrosses average ?75% B alleles and
25% S alleles).
Single crosses. Two comparisons were made for each of the eight
backcrosses. Specifically, each backcross was compared with 1) the
Table 1. The eight B-S-derived backcrosses
Group B Backcrosses (yielded B-B or B-S alleles)Group S Backcrosses (yielded S-S or B-S alleles)
B damB sire S dam S sire
Cross 4: B ? (B ? S)F1 ? B.BS
Cross 5: B ? (S ? B)F1 ? B.SB
Cross 2: (B ? S)F1 ? B ? BS.B
Cross 7: (S ? B)F1 ? B ? SB.B
Cross 9: S ? (B ? S)F1 ? S.BS
Cross 10: S ? (S ? B)F1 ? S.SB
Cross 3: (B ? S)F1 ? S ? BS.S
Cross 8: (S ? B)F1 ? S ? SB.S
B, C57BL/6J strain; S, 129X1/SvJ strain. Crosses 1 and 6 were assigned to different crosses being generated at the same time as these backcrosses and were
not part of this study.
CONFIRMATION OF MAJOR LOCI FOR HALI SURVIVAL TIME
Physiol Genomics • VOL 38 • www.physiolgenomics.org
8. Broman KW, Sen S, Owens SE, Manichaikul A, Southard-Smith EM,
Churchill GA. The X chromosome in quantitative trait locus mapping.
Genetics 174: 2151–2158, 2006.
9. Broman KW, Wu H, Sen S, Churchill GA. R/qtl: QTL mapping in
experimental crosses. Bioinformatics 19: 889–890, 2003.
10. Carraway MS, Suliman HB, Kliment C, Welty-Wolf KE, Oury TD,
Piantadosi CA. Mitochondrial biogenesis in the pulmonary vasculature
during inhalational lung injury and fibrosis. Antioxid Redox Signal 10:
11. Chow CW, Herrera Abreu MT, Suzuki T, Downey GP. Oxidative
stress and acute lung injury. Am J Respir Cell Mol Biol 29: 427–431,
12. Crapo JD, Barry BE, Foscue HA, Shelburne J. Structural and biochem-
ical changes in rat lungs occurring during exposures to lethal and adaptive
doses of oxygen. Am Rev Respir Dis 122: 123–143, 1980.
13. De Sanctis GT, Merchant M, Beier DR, Dredge RD, Grobholz JK,
Martin TR, Lander ES, Drazen JM. Quantitative locus analysis of
airway hyperresponsiveness in A/J and C57BL/6J mice. Nat Genet 11:
14. de Torres JP, Cote CG, Lopez MV, Casanova C, Diaz O, Marin JM,
Pinto-Plata V, de Oca MM, Nekach H, Dordelly LJ, Aguirre-Jaime A,
Celli BR. Gender differences in mortality in patients with COPD. Eur
Respir J 33: 528–535, 2009.
15. Ferring M, Vincent JL. Is outcome from ARDS related to the severity of
respiratory failure? Eur Respir J 10: 1297–1300, 1997.
16. Ganguly K, Stoeger T, Wesselkamper SC, Reinhard C, Sartor MA,
Medvedovic M, Tomlinson CR, Bolle I, Mason JM, Leikauf GD,
Schulz H. Candidate genes controlling pulmonary function in mice:
transcript profiling and predicted protein structure. Physiol Genomics 31:
17. Gharaee-Kermani M, Hatano K, Nozaki Y, Phan SH. Gender-based
differences in bleomycin-induced pulmonary fibrosis. Am J Pathol 166:
18. Gunther A, Walmrath D, Grimminger F, Seeger W. Pathophysiology
of acute lung injury. Semin Respir Crit Care Med 22: 247–258, 2001.
19. Hager R, Cheverud JM, Wolf JB. Maternal effects as the cause of
parent-of-origin effects that mimic genomic imprinting. Genetics 178:
20. Jin Y, Kim HP, Chi M, Ifedigbo E, Ryter SW, Choi AM. Deletion of
caveolin-1 protects against oxidative lung injury via up-regulation of heme
oxygenase-1. Am J Respir Cell Mol Biol 39: 171–179, 2008.
21. Kleeberger SR, Reddy S, Zhang LY, Jedlicka AE. Genetic susceptibil-
ity to ozone-induced lung hyperpermeability: role of Toll-like receptor 4.
Am J Respir Cell Mol Biol 22: 620–627, 2000.
22. Malkinson AM, Radcliffe RA, Bauer AK. Quantitative trait locus mapping
of susceptibilities to butylated hydroxytoluene-induced lung tumor promotion
and pulmonary inflammation in CXB mice. Carcinogenesis 23: 411–417,
23. Matesic LE, De Maio A, Reeves RH. Mapping lipopolysaccharide
response loci in mice using recombinant inbred and congenic strains.
Genomics 62: 34–41, 1999.
24. Matthay MA, Bhattacharya S, Gaver D, Ware LB, Lim LH,
Syrkina O, Eyal F, Hubmayr R. Ventilator-induced lung injury:
in vivo and in vitro mechanisms. Am J Physiol Lung Cell Mol Physiol
283: L678–L682, 2002.
25. Matthay MA, Zimmerman GA. Acute lung injury and the acute respi-
ratory distress syndrome: four decades of inquiry into pathogenesis and
rational management. Am J Respir Cell Mol Biol 33: 319–327, 2005.
26. Montgomery AB, Stager MA, Carrico CJ, Hudson LD. Causes of
mortality in patients with the adult respiratory distress syndrome. Am Rev
Respir Dis 132: 485–489, 1985.
27. Ohtsuka Y, Wang XT, Saito J, Ishida T, Munakata M. Genetic linkage
analysis of pulmonary fibrotic response to silica in mice. Eur Respir J 28:
28. Pagano A, Donati Y, Metrailler I, Barazzone Argiroffo C. Mitochon-
drial cytochrome c release is a key event in hyperoxia-induced lung injury:
protection by cyclosporin A. Am J Physiol Lung Cell Mol Physiol 286:
29. Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, Singh JM,
Scales DC, Stather DR, Li A, Jones A, Gattas DJ, Hallett D, Tomlin-
son G, Stewart TE, Ferguson ND. Has mortality from acute respiratory
distress syndrome decreased over time?: a systematic review. Am J Respir
Crit Care Med 179: 220–227, 2009.
30. Pieczenik SR, Neustadt J. Mitochondrial dysfunction and molecular
pathways of disease. Exp Mol Pathol 83: 84–92, 2007.
31. Postma DS. Gender differences in asthma development and progression.
Gend Med 4, Suppl B: S133–S146, 2007.
32. Prows DR, Hafertepen AP, Winterberg AV, Gibbons WJ Jr, Liu CY,
Nick TG. Genetic analysis of hyperoxic acute lung injury survival in
reciprocal intercross mice. Physiol Genomics 30: 271–281, 2007.
33. Prows DR, Hafertepen AP, Winterberg AV, Gibbons WJ Jr, Nick TG.
A genetic mouse model to investigate hyperoxic acute lung injury sur-
vival. Physiol Genomics 30: 262–270, 2007.
34. Prows DR, Leikauf GD. Quantitative trait analysis of nickel-induced
acute lung injury in mice. Am J Respir Cell Mol Biol 24: 740–746, 2001.
35. Ratner V, Starkov A, Matsiukevich D, Polin RA, Ten VS. Mitochondrial
dysfunction contributes to alveolar developmental arrest in hyperoxia-ex-
posed mice. Am J Respir Cell Mol Biol 40: 511–518, 2009.
36. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M,
Stern EJ, Hudson LD. Incidence and outcomes of acute lung injury.
N Engl J Med 353: 1685–1693, 2005.
37. Schapira AH. Mitochondrial disease. Lancet 368: 70–82, 2006.
38. Sidak Z. Rectangular confidence region for the means of multivariate
normal distributions. J Am Stat Assoc 62: 626–633, 1967.
39. Sloane PJ, Gee MH, Gottlieb JE, Albertine KH, Peters SP, Burns JR,
Machiedo G, Fish JE. A multicenter registry of patients with acute
respiratory distress syndrome. Physiology and outcome. Am Rev Respir
Dis 146: 419–426, 1992.
40. Suchyta MR, Clemmer TP, Elliott CG, Orme JF Jr, Weaver LK. The
adult respiratory distress syndrome. A report of survival and modifying
factors. Chest 101: 1074–1079, 1992.
41. Van Winkle LS, Gunderson AD, Shimizu JA, Baker GL, Brown CD.
Gender differences in naphthalene metabolism and naphthalene-induced
acute lung injury. Am J Physiol Lung Cell Mol Physiol 282: L1122–
42. Vancza EM, Galdanes K, Gunnison A, Hatch G, Gordon T. Age,
strain, and gender as factors for increased sensitivity of the mouse lung to
inhaled ozone. Toxicol Sci 107: 535–543, 2009.
43. Vincent JL, Zambon M. Why do patients who have acute lung injury/
acute respiratory distress syndrome die from multiple organ dysfunction
syndrome? Implications for management. Clin Chest Med 27: 725–731,
44. Ward NS, Waxman AB, Homer RJ, Mantell LL, Einarsson O, Du Y,
Elias JA. Interleukin-6-induced protection in hyperoxic acute lung injury.
Am J Respir Cell Mol Biol 22: 535–542, 2000.
45. Ward PA. Acute lung injury: how the lung inflammatory response works.
Eur Respir J Suppl 44: 22s–23s, 2003.
46. Ward PA, Mulligan MS. Molecular mechanisms in acute lung injury.
Adv Pharmacol 24: 275–292, 1993.
47. Waxman AB, Einarsson O, Seres T, Knickelbein RG, Warshaw JB,
Johnston R, Homer RJ, Elias JA. Targeted lung expression of interleu-
kin-11 enhances murine tolerance of 100% oxygen and diminishes hyper-
oxia-induced DNA fragmentation. J Clin Invest 101: 1970–1982, 1998.
48. Wesselkamper SC, Chen LC, Gordon T. Quantitative trait analysis of
the development of pulmonary tolerance to inhaled zinc oxide in mice.
Respir Res 6: 73, 2005.
49. Westfall P, Tobias R, Rom D, Wolfinger R, Hochberg Y. Multiple
Comparisons and Multiple Tests Using SAS. Cary, NC: SAS Institute,
50. Zambon M, Vincent JL. Mortality rates for patients with acute lung
injury/ARDS have decreased over time. Chest 133: 1120–1127, 2008.
51. Zilberberg MD, Epstein SK. Acute lung injury in the medical ICU:
comorbid conditions, age, etiology, and hospital outcome. Am J Respir
Crit Care Med 157: 1159–1164, 1998.
CONFIRMATION OF MAJOR LOCI FOR HALI SURVIVAL TIME
Physiol Genomics • VOL 38 • www.physiolgenomics.org