Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Cryopreservation of human oocytes: a review of current problems and perspectives

It is well recognized that the ability to cryopreserve unfertilizedhuman oocytes would make a significant contribution to infertilitytreatment. However, despite considerable interest, very fewsuccessful pregnancies have arisen from cryopreserved oocytesafter thawing, insemination and transfer of the subsequent embryo.The reasons for this lack of progress may well result from adearth of information on how the various biophysical changesduring a cryopreservation regimen affect human oocyte function.Recently, fundamental studies on the effects of cooling, membranepermeability, cryoprotectant addition and ice formation havebeen performed on human oocytes by a number of groups, and theseform the basis of the current review. It is likely that successfulhuman oocyte cryopreservation will only follow once these factorsare fully understood, but the existing base of knowledge shouldprovide a platform for further improvements in the techniquescurrently employed.     

Testing of the stability of intensity modulated beams generated with dynamic multileaf collimation, applied to the MM50 racetrack microtron

Recently, we have published a method for the calculation of required leaf trajectories to generate optimized intensity modulated x-ray beams by means of dynamic multileaf collimation [Phys. Med. Biol. 43, 1171-1184 (1998)]. For the MM50 Racetrack Microtron it has been demonstrated that the dosimetric accuracy of this method, in combination with the dose calculation algorithm of the Cadplan 3D treatment planning system, is adequate for a clinical application (within 2% or 0.2 cm). Prior to initiating patient treatment with dynamic multileaf collimation (DMLC), tests have been performed to investigate the stability of DMLC fields generated at the MM50, (i) in time, (ii) subject to gantry rotation and (iii) in case of treatment interrupts, e.g., caused by an error detected by the treatment machine. The stability of relative dose profiles, normalized to a reference point in a relatively flat part of the modulated beam profile, was assessed from measurements with an electronic portal imaging device (EPID), with a linear diode array attached to the collimator and with film. The dose in the reference point was monitored using an ionization chamber. Tests were performed for several intensity modulated fields using 10 and 25 MV photon beams. Based on film measurements for sweeping 0.1 cm leaf gaps it was concluded that in an 80 days period the variation in leaf positioning was within 0.05 cm, without requiring any recalibration. For a uniform 10x10 cm2 field, realized dynamically by a scanning 0.4x10 cm2 slit beam, a maximum variation in slit width of 0.01 cm was derived from ionization chamber measurements, both in time and for gantry rotation. For a clinical example, the dose in the reference point reproduced within 0.2% (1 SD) over a period of 100 days. Apart from regions with very large dose gradients, variations in the relative beam profiles measured with the EPID were generally less than 1% (1 SD). For different gantry angles the dose profiles also reproduced within 1%, showing that gravity has a negligible influence. No significant deviations between uninterrupted and interrupted treatments could be observed, indicating that the effects of acceleration and deceleration of the leaves are negligible and that a DMLC treatment can be finished correctly after a treatment interrupt. Our previous and present studies have demonstrated that the dosimetric accuracy and stability of intensity modulated beams, generated at the MM50 by means of dynamic multileaf collimation, are adequate for clinical use. Patient treatment using dynamic multileaf collimation has been started in our clinic.