A humanized mouse model for the reduced folate carrier
Section snippets
Isolation and characterization of a P1 clone containing the hRFC gene
A human genomic P1 BAC library [38] was screened as previously described [4], [16] using standard PCR conditions with a pair of oligonucleotide primers which amplify hRFC. The forward primer was 5′-CTCCTTCTCCACGCTCAAC and the reverse primer was 5′-GAAGCCGAGGTTTCGCACC. These primers produce an amplicon of 135 nt from positions 350–484 of the second coding exon of the hRFC gene. A P1 clone designated P33A12 (77.4 kb insert in 16 kb vector pAd10SacBII), containing 33.2 kb of the hRFC gene and
Isolation and characterization of a P1 clone containing a functional hRFC gene
The P1 clone P33A12 was isolated as described in Materials and methods. The insert of P33A12 spans from nucleotide (nt) 45,714,910 to 45,792,261 of chromosome 21, and is 77,351 nt long. P33A12 contains 9960 nt of DNA 5′ (telomeric) of the translation start codon of hRFC and 44,157 nt 3′ of the hRFC 3′ UTR, and does not contain any other functional genes or hypothetical genes.
To test whether the hRFC gene in P33A12 is functional, we transfected HeLa R5 cells, which are null for the hRFC gene [50]
Discussion
In this manuscript, we describe production and characterization of transgenic mice containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in the TghRFC1 mice by qPCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues whereas, as expected, the tissue specificity
Acknowledgments
We thank Dr. I. David Goldman (Albert Einstein School of Medicine) for his gifts of hRFC-null R5 HeLa cells and C57BL/6 mice with one active and one inactive mRFC allele, and Jean M. Smith and Lynne Meltesen (Colorado Genetics Laboratory, Department of Pathology, University of Colorado at Denver and Health Sciences Center) for FISH analysis of metaphase spreads of transgenic mice. DNA sequencing was performed at the University of Colorado Cancer Center DNA Sequencing & Analysis Core. This study
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