Human artificial chromosome (HAC) vectors are an important gene transfer system for expression and complementation studies. We describe a significant advance in HAC technology using infectious herpes simplex virus type 1 (HSV-1) amplicon vectors for delivery. This highly efficient method has allowed geneexpressing HACs to be established in glioma-, kidney-and lungderived cells. We also developed an HSV-1 hypoxanthine phosphoribosyltransferase (HPRT) HAC vector, which generated functional HPRT-expressing HACs that complemented the genetic deficiency in human cells. The transduction efficiency of the HSV-1 HAC amplicons is several orders of magnitude higher than lipofection-mediated delivery. Studies on HAC stability between cell types showed important differences that have implications for HAC development and gene expression in human cells. This is the first report of establishing gene-expressing HACs in human cells by using an efficient, high-capacity viral vector and by identifying factors that are involved in cell-typespecific HAC instability. The work is a significant advance for HAC technology and the development of HAC gene expression systems in human cells.
The patient was referred for paediatric opinion because at the age of 13 years she was still only I25 cm. tall. However, her secondary sexual development was normal and she first menstruated shortly after her I3th birthday. After a few months without periods she began to menstruate regularly. Two buccal smears taken at this time failed to show sex chromatin, and it seemed possible, that despite her normal secondary sexual development and regular menstruation, she might have a sex chromosome abnormality. She was referred for detailed chromosomal study.Received May I4, I965.Sex Chromatin and Chromosome Analysis. Of the I20 nuclei examined in buccal smear preparations, 6 (5%) had a single, normal-sized, sex chromatin body.Sex chromatin bodies are found by this observer (A.M.B.) with a frequency of 25-60% in normal females and are not observed in normal males. Further sex chromatin preparations were obtained from skin culture. These latter preparations were made in parallel with a control culture from a normal female. The preparations derived from the normal female were sex chromatin positive, but of 300 cells examined in the skin culture derived from the propositus, not one showed a sex chromatin body. A blood smear preparation was analysed and 3 drumsticks (o-6%) were found in 500 polymorphonuclear leucocytes. Mittwoch (i964) in an investigation of I2 normal females found a mean drumstick count of 3-66%.Chromosome counts showed two cell types with 45 and 46 chromosomes, in three independent blood cultures and a skin culture.Analysis demonstrated XO and XO plus a ring chromosome karyotypes (Table I). The ring chromosome was of variable size (Fig. i).The pattern of DNA synthesis was studied in the third blood culture, labelled for 4 hours with tritiated thymidine (Bishop and Bishop, I963). In all, 300 cells were examined: 4I had a count of 46 chromosomes. I4 of these 4I were labelled, and 8 showed a concentration of labelling over the ring chromosome with slight labelling over the other members of the complement (Fig. 2). These 8 cells were presumably at the end of DNA synthesis when labelling occurred. It was, therefore, assumed that the ring chromosome was a structurally abnormal, late-labelling, X chromosome. A
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