Molecular cloning of feline CD34.
In humans, baboons, dogs and mice CD34 is a cell surface molecule that is expressed on primitive hematopoietic cells and in all these species CD34 positive cells can be used to effect long-term haematopoietic reconstitution. CD34 positive haematopoietic cells therefore provide a convenient and relatively small cell population to target when attempting gene therapy via the haematopoietic system. In order to develop the mucopolysaccharidosis type VI (MPS VI) cat as a model for haematopoietic cell-mediated gene therapy we have isolated the feline CD34 gene as a first step in the generation of antibodies for purification of feline CD34 positive cells. The coding sequence for feline CD34 was isolated from brain cDNA using the polymerase chain reaction (PCR) with oligonucleotides designed to conserved regions of known CD34 gene sequences as primers. Sequence analysis of PCR products revealed the complete amino acid sequence of feline CD34 and allowed analysis of sequence conservation with CD34 from other species. Northern blot analysis showed a 2.6 kb CD34 transcript was present in feline brain, spleen, heart, testis and thymus, and to a lesser extent, in liver. A full-length cDNA clone of the feline CD34 coding sequence was assembled and expressed in CHO-K1 cells. The isolation and expression of the feline CD34 cDNA should facilitate the production of antibodies suitable for the purification of CD34 positive cells.
Yogalingam
Gouri
G
Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, SA 5006, Australia.
Anson
Donald S
DS
D000595:Amino Acid Sequence; D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002415:Cats / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D006224:Cricetinae; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D009087:Mucopolysaccharidosis VI / Q000276:immunology*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D018952:Antigens, CD34; D012313:RNA