Search

We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.Copyright 1993, 1999 Academic Press

The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitrotranscription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situhybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5′ or 3′ end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitrotranscription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situhybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.

Condensed molasses fermentation solubles (CMS), an effluent from the production of lysine, was evaluated as a nonprotein nitrogen supplement for ruminants by measuring the availability of its nitrogen to rumen microorganisms grown in batch cultures and by comparing CMS to urea as a source of supplemental nitrogen for growing cattle. In vitro dry matter digestion studies showed that, with 1 ml or less of rumen inoculum, microbial digestion was enhanced more (P< .05) by the addition of CMS than by the addition of urea to 100 mg of cellulose. These stimulatory effects of CMS were absent when either the amount of inoculum (5.0 ml) or cellulose (250 mg) was increased and when wheat straw or alfalfa replaced cellulose as the substrate. Growth rate and feed intake for cattle fed a high-cob/cracked-corn diet containing 2.5 or 5.0% CMS were lower (P< .05) than for cattle fed the control diet containing urea. Digestibility of dry matter, crude protein, neutral detergent fiber and acid detergent fiber were reduced (P< .05) by the addition of CMS. Addition of CMS also decreased feed utilization, although the differences were not statistically significant. In conclusion, the nitrogen in CMS was available to rumen microorganisms growth in batch culture; however, CMS was not satisfactory as a substitute for all the urea in a diet for growing cattle containing over 45% of dietary N from the supplemental N source.

Prednisone inhibits bone formation and causes bone loss. To investigate possible mechanisms and sites, the effects of sham operation, ovariectomy, and prednisone were determined on bone and mineral metabolism in 7-week-old growing female rats. Forty animals were divided into groups of 10 each. Sham operation and ovariectomy were performed. One week later, pellets containing 5 mg prednisone or drug free were implanted S.C. at the back of the neck. Four weeks later, animals were sacrificed and tibiae were removed for histomorphometric analysis of the middiaphysis and proximal metaphysis. In both sham-operated and ovariectomized rats, prednisone (1) reduced weight gain (P<0.02) and did not alter uterine weight; (2) lowered serum magnesium (Mg) (P<0.001) and did not change serum calcium (Ca), phosphate (P), 25-hydroxyvitamin D (25OHD), or 1,25-dihydroxyvitamin D [1,25(OH)2D]; (3) produced striking increases in calcified cartilage, reduced cross-sectional area (P<0.05) and cortical area (P<0.01) and did not change medullary area of the tibial diaphysis; (4) lowered periosteal and endocortical bone formation and apposition rates; and (5) increased mean cancellous bone area (P<0.05) and cancellous bone perimeter (P<0.01) of the tibial metaphysis. In both control and prednisone-treated rats, ovariectomy (1) reduced uterine weight (P<0.001); (2) did not change serum Ca, P, Mg, 25OHD, or 1,25(OH)2D; (3) did not change mean cross-sectional, medullary, or cortical areas; (4) increased periosteal bone formation and apposition rates (P<0.01) and did not alter endosteal bone formation and apposition rates, and (5) decreased cancellous bone area (P<0.01) and cancellous bone perimeter (P<0.01). Thus, in short-term studies, prednisone increased calcified cartilage and inhibited the formation of cortical bone at periosteal and endosteal surfaces and reduced cortical bone of the tibia in both sham-operated and ovariectomized, rapidly growing animals.

Abstract.: Leukemia inhibitory factor (LIF) is a single-chain polypeptide that previously was shown in mice to produce hypercalcemia and influence skeletal growth and turnover. We performed dose-response studies to determine if LIF alters the serum calcium or histomorphometry of the tibia in growing male rats. Forty animals were divided into five groups of eight animals each. Recombinant human LIF, 0.01, 0.1, 1, or 10 μg/100 g body wt, or vehicle was administered daily S.C. for 3 weeks. Compared with controls it was found that LIF increased mean serum calcium at the two highest doses (11.4 ± 0.1 versus 10.8 ± 0.1 mg/dl, P= 0.0005 by one-way analysis of variance (ANOVA) but did not alter static or dynamic measurements of histomorphometry or length of the tibia. We conclude that in growing rats, high systemic concentrations of LIF result in hypercalcemia with no changes in bone turnover.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.