Application of functional genomics to the chimeric mouse model of HCV infection: optimization of microarray protocols and genomics analysis.
BACKGROUND
Many model systems of human viral disease involve human-mouse chimeric tissue. One such system is the recently developed SCID-beige/Alb-uPA mouse model of hepatitis C virus (HCV) infection which involves a human-mouse chimeric liver. The use of functional genomics to study HCV infection in these chimeric tissues is complicated by the potential cross-hybridization of mouse mRNA on human oligonucleotide microarrays. To identify genes affected by mouse liver mRNA hybridization, mRNA from identical human liver samples labeled with either Cy3 or Cy5 was compared in the presence and absence of known amounts of mouse liver mRNA labeled in only one dye.


RESULTS
The results indicate that hybridization of mouse mRNA to the corresponding human gene probe on Agilent Human 22 K oligonucleotide microarray does occur. The number of genes affected by such cross-hybridization was subsequently reduced to approximately 300 genes both by increasing the hybridization temperature and using liver samples which contain at least 80% human tissue. In addition, Real Time quantitative RT-PCR using human specific probes was shown to be a valid method to verify the expression level in human cells of known cross-hybridizing genes.


CONCLUSIONS
The identification of genes affected by cross-hybridization of mouse liver RNA on human oligonucleotide microarrays makes it feasible to use functional genomics approaches to study the chimeric SCID-beige/Alb-uPA mouse model of HCV infection. This approach used to study cross-species hybridization on oligonucleotide microarrays can be adapted to other chimeric systems of viral disease to facilitate selective analysis of human gene expression.
Walters
Kathie-Anne
KA
Department of Microbiology, University of Washington, Seattle, WA, USA. kathiw@u.washington.edu
Joyce
Michael A
MA
Thompson
Jill C
JC
Proll
Sean
S
Wallace
James
J
Smith
Maria W
MW
Furlong
Jeff
J
Tyrrell
D Lorne
DL
Katze
Michael G
MG
D000818:Animals; D002678:Chimera / Q000821:virology*; D019295:Computational Biology / Q000379:methods; D004195:Disease Models, Animal; D015870:Gene Expression / Q000502:physiology; D023281:Genomics / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D050356:Lipid Metabolism / Q000235:genetics; D008099:Liver / Q000502:physiology* / Q000821:virology*; D051379:Mice; D016513:Mice, SCID; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D013389:Succinate-CoA Ligases / Q000235:genetics
D012313:RNA; D013389:Succinate-CoA Ligases