Not shown is the adapter primer that is ligated to each end of the cDNA, to which oligonucleotide AP1 is complementary. R series denotes oligonucleotides with sequence unique to the rhesus macaque factor IX cDNA sequence. H series denotes oligonucleotides completely homologous to human factor IX coding sequence. (Below) Schematic diagram of rhesus macaque factor IX cDNA depicting the position of oligonucleotide primers used to amplify overlapping RACE PCR fragments and determine the nucleic acid sequence. Note the double bands observed for 3′ RACE PCR fragments 3′2 and 3′4, due to the two different polyadenylation signal sequences, separated by 224 base pairs in the cDNA. Lane 5′8: amplimer obtained with AP1 and H8 primers lane 5′9: amplimer obtained with AP1 and H9 primers lane 5′10: amplimer obtained with AP1 and H10 primers lane 3′2: amplimer obtained with H2 and AP1 primers lane 3′4: amplimer obtained with H4 and AP1 primers. ![]() (Above) 1% agarose gel electrophoresis of 5′ and 3′ RACE PCR fragments obtained by amplification of adapter-ligated first-strand DNA template with adapter primer AP1 (Clonetech) and various factor IX–specific oligonucleotides. Strategy for determination of the nucleic acid sequence of the rhesus macaque factor IX cDNA. 5′ and 3′ rapid amplification of cDNA ends (RACE). Polyadenylated RNA was used for first-strand DNA synthesis using reverse transcriptase and second-strand synthesis with Klenow DNA polymerase, after which the double-stranded DNA fragment was ligated to adapter-primers (Clontech, Palo Alto, CA) and subjected to polymerase chain reaction (PCR) amplification using gene-specific oligonucleotide primers and primers specific for the adapter ligated to the double-stranded DNA. Total RNA was stored under isopropyl alcohol at −20☌ until polyadenylated RNA was purified with the Promega (Madison, WI) Polyattract kit. Frozen liver was disrupted with an automated tissue homogenizer, and total RNA was prepared by extraction with Trizol (Life Sciences, Bethesda, MD) according to manufacturer’s directions. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.įresh liver obtained from a female rhesus after euthanasia for an unrelated study was flash frozen in dry ice and stored under liquid nitrogen before isolation of total RNA for cloning of the factor IX cDNA. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody–purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. ![]() We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). ![]() The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. The cDNA has a large 3′ untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA.
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