A new supplemented medium has been developed to improve human amniotic fluid cell growth and to reduce the dependence on exogenously added serum. The medium consists of a mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with Hepes, antibiotics, and 10 growth-promoting factors at 4% fetal bovine serum. Good clonal growth is achieved consistently in 8-13 days and is associated with large numbers of metaphase cells. Primary clones may be analyzed directly, thereby reducing difficulty with interpretation of chromosomal mosaicism. This medium could also be used for cultivation of fetal solid tissues and peripheral blood cultures of lymphocytes.Numerous hormones have been detected in human amniotic fluid-such as human growth hormone, human follicle-stimulating hormone, human luteinizing hormone, human chorionic gonadotropin, thyroid hormones, insulin, glucagon, testosterone, progesterone, estradiol, and cortisone, among others. The source and entry of some hormones, their concentrations, and their possible physiological role in pregnancy have been reviewed by Dawood (1).Most prenatal genetic diagnostic studies rely on amniocentesis and the subsequent growth of human amniotic fluid cells (HAFC). Despite various techniques used in HAFC cultures, fetal bovine serum (FB serum) is perhaps one of the most important determinants in achievement of cell growth in vitro. A level of 15-30% FB serum is most often added to HAFC cultures in a wide range of chemically defined nutrient media (2-5). The high concentration of the serum may increase the chance of mycoplasma contamination, which is known to cause structural aberrations in chromosomes prepared from HAFC cultures (6). It may also exhibit an inhibitory effect on cell growth (7).The studies described here were -undertaken to determine whether a partial supplement of some pregnancy-related hormones, originally present in amniotic fluid, to a nutrient medium would lower serum requirements of HAFC. In addition, experiments were designed to determine if a supplement of growth additives would minimize vast variations in growth stimulation properties in commercially prepared serum used for tissue culture purposes. By a modification of serum-free culture systems, a medium supplemented with 10 growth-promoting factors at 4% FB serum consistently resulted in good clonal growth in 8-13 days. In conjunction with the use of chamber/ slides, chromosomal analysis from primary culture in situ could be performed in a relatively short time.MATERIALS AND METHODS Culture Medium. Coon's modification of Ham's F12 medium (Coon's F12 medium) was routinely used in our prenatal diagnostic laboratory; therefore, it was included as a control medium for various growth assays (8).Unless otherwise stated, the basic culture medium [serumfree (SF) medium] was a 1:1 mixture of Dulbecco-Vogt modified Eagle's medium (DVME medium) and Ham's F12 medium (F12 medium) supplemented with 15 mM Hepes and 1.2 g of NaHCO3, 40 mg of penicillin, 8 mg of ampicillin, and 90 mg of stre...
Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.
Reduction in serum requirement for culture of primary human amniotic fluid cells can be achieved by the addition of 10 growth-promoting factors to the nutrient medium. This supplemented medium preserves cell types normally found in amniotic fluid cell cultures supplemented with 20-30 per cent fetal bovine serum. The volume of amniotic fluid required to initiate culture can be as little as 1 ml. Amniotic fluid samples contaminated with red blood cells with no visible clot also grow well in the low serum medium. Cell-free amniotic fluid combined with equal parts of supplemented medium is useful in initiating cell culture.
In vitro characteristics of human fetal cells have been investigated after chorionic villus sampling at the first trimester and amniocentesis at the second trimester of pregnancy. Light microscopy revealed heterogeneous morphology of cell types in both the chorionic villus culture and the amniotic fluid cultures. Based on the experiments performed, chorionic villus cells are more sensitive to pronase, trypsin, and versene during subculture and have a higher DNA content per single cell and release more [125I]-Beta-human chorionic gonadotropin into culture medium than those found in amniotic fluid cells. The practical applications of this study are discussed.
SUMMARY: The procedure for human gene mapping by in situ hybridization is described. By using specific radiolabeled probes, specific homologous sequences can be localized as silver grains on chromosome spreads of cytogenetic preparations. It is therefore possible to obtain genetic information on translocation, breakpoint, deletion, amplification, and rearrangement at the chromosomal level of many human diseases.
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