Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors.
We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.
Brown
O A
OA
Department of Medicine, University of Manchester School of Medicine, UK.
Santer
R M
RM
Shering
A F
AF
Larregina
A T
AT
Morelli
A E
AE
Southgate
T D
TD
Castro
M G
MG
Lowenstein
P R
PR
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D003673:Defective Viruses / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D007421:Intestine, Small / Q000201:enzymology / Q000294:innervation*; D009474:Neurons / Q000201:enzymology; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001616:beta-Galactosidase