Gene targeting in adult rhesus macaque fibroblasts

<p>Abstract</p> <p>Background</p> <p>Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology.</p> <p>Results</p> <p>A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term <it>in vitro </it>manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer.</p> <p>Conclusion</p> <p>The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.</p

Comparison of Two Serologic Tests for the Diagnosis of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

The sensitivity and specificity of the virus neutralization (VN) and indirect immunofluorescence (IF A) tests for the detection of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) was compared using 1,605 field sera from pigs with reproductive and/or respiratory disease. In the VN test, sera were diluted 2-fold (1:4 to 1:512); incubated with an equal volume of 100 TCID50 of PRRSV (ATCC VR-2332); and then the mixtures were added to duplicate wells of CL2621 cells. The VN titer was determined to be the highest dilution of sera which blocked cytopathic effects in CL262 l cells. In the IF A, CL2621 cells in 96-well microtiter plates were inoculated with PRRSV and cells were fixed 24 h later with 80% acetone. Sera were diluted 4-fold (1 :5 to 1:5, 120), each dilution was added to duplicate wells of inoculated and uninoculated CL262 l cells, and the sera containing antibodies were detected using FITC-labeled rabbit anti-swine IgG. Fifty percent (69/139) of the herds tested had animals with antibodies to VR-2332 by both VN and IFA. Thirty-two and 3% of all sera were positive by IF A and VN, respectively. Seven percent of all samples were positive by both assays; 58% were negative by both assays; and 8% (27/326) of the samples had antibody to both the European and North American isolates of PRRSV. Thirty-eight percent (297/776) of the samples from sows and gilts and 7% (9/129) of boars tested positive by VN and IPA Nineteen percent (96/501) of the breeding and 6% (15/260) of the finishing pigs were positive for PRRSV antibodies. In herds with 3 7 to 100 animals, 42% (16/38) tested positive, while 44% (24/54) of herds with more than 200 pigs were positive by VN and IF A Specificities of VN and IF A were calculated to be 97% and 86%, and sensitivities were 25% and 44%, respectively. Statistically, the IFA was significantly more effective than VN in detecting PRRSV antibodies