Suppression of Mamu-AG by RNA interference.
OBJECTIVE
The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu-AG, the rhesus monkey homology of human leukocyte antigen (HLA)-G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.


METHODS
A series of potential target short hairpin RNA (shRNA) sequences to suppress Mamu-AG expression was screened, which identified an optimal sequence to use in transfection experiments. Knockdown in two different Mamu-AG-expressing cell lines was measured by flow cytometry. Cytotoxicity assays were performed to correlate Mamu-AG expression with NK cell cytotoxicity.


RESULTS
Decreased expression of Mamu-AG by short interfering RNA (siRNA) (70-80%) in cell types tested was associated with increased lysis of Mamu-AG target cells.


CONCLUSIONS
Target sequences have been identified that knocked down Mamu-AG expression by RNAi and increased lysis by NK cells. This supports the concept that NK cell receptors recognize this placental non-classical MHC class I molecule.
Drenzek
Jessica G
JG
Department of Obstetrics and Gynecology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53715-1299, USA.
Vidiguriene
Jolanta
J
Vidiguris
Geminis
G
Grendell
Richard L
RL
Dambaeva
Svetlana V
SV
Durning
Maureen
M
Golos
Thaddeus G
TG
D000818:Animals; D002460:Cell Line; D005260:Female; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007111:Immunity, Cellular / Q000235:genetics / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D008253:Macaca mulatta; D010920:Placenta / Q000276:immunology* / Q000378:metabolism; D011247:Pregnancy; D034622:RNA Interference
D015395:Histocompatibility Antigens Class I