Genetic Engineering & Biotechnology News

NOV15 2017

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Genetic Engineering & Biotechnology News | GENengnews.com | NOVEMBER 15, 2017 | 13 analytical approach provides a direct mea- sure of a chimera's appropriateness for breed- ing to attain the desired genotype efficiently. In parallel, it is very useful to analyze the fertilization potential of the cryopreserved sperm sample by using an aliquot in a test IVF. Knowing which male is the most fer- tile and which has the highest likelihood of transmitting the ES cell genome to the next generation facilitates well-informed choices for cohort production. If the results indicate the clone(s) used in the blastocyst injection have poor germline potential and/or result in infertile or sub-fertile males, one can quickly select additional clones for chimera genera- tion. If male chimeras with good germline potential and fertility are identified, one can move forward with greater confidence that the cohort will meet the study needs. Speeding Cohort Delivery After chimeras have been chosen for breeding, IVF can speed production of a study-size cohort considerably. The selected male's sperm can be used to simultaneously fertilize hundreds of oocytes, generating many offspring faster and more predictably than with natural mating. IVF has other ad- vantages: It occurs on a more predictable timeline, making it easier to plan for studies, and several rounds of chimera breeding can be skipped, saving three to four months. IVF also offers the flexibility to produce, then breed, mice at the most appropriate health status for the study. To maximize the speed advantages pro- vided by the strategy outlined above, it is advantageous to produce male chimeras without a selectable marker cassette, avoid- ing the need to delete the selection marker later through additional breeding. This can be accomplished through in vivo deletion in the male germ line using self-excising selec- tion cassettes, or use of CRISPR in ES cells to eliminate the need for a selectable marker. Alternatively, in vitro deletion of a standard selection cassette in ES cells by recombinase transfection can be employed, but at the ex- pense of some added time and effort. Lastly, if these strategies are not feasible, one could use oocytes derived from a recombinase- expressing mouse line for the IVF. However, in this case the deleter transgene or knock-in allele would need to be selected against dur- ing breeding. Results in Action Data from a real-world custom model generation project demonstrates the advan- tages of the strategies outlined above. As illustrated in Figure 2, based on coat color alone, chimeras #1 and/or #4 might have been selected for breeding, but molecu- lar analysis revealed chimera #4 would be the better choice of the two. While chimeras #3 and #6 would likely not have been consid- ered for breeding, molecular analysis indicat- ed that both would be reasonable choices. In this case, chimera #2 was selected, based on the combination of a high fertility rate and high percentage of the modified allele in the germ line, and used in an IVF with oocytes derived from wild-type females. All 79 pups born were black, indicating 100% germline transmission of the ES cells. Of the 72 mice genotyped, 34 (47%) were heterozygous, which was consistent with expectations. Sperm genotyping for selecting breed- ers, combined with IVF for rapid cohort expansion, is a rational approach to mouse model generation and breeding for complex genome modification projects requiring an ES cell-based targeting approach (Figure 3). This strategy can assist investigators in ad- vancing preclinical studies sooner, making data-driven strategic decisions, and shorten- ing drug discovery timelines. CORe ™ M ini Bioreactor POWER FOR DISCOVERY Culture Optimization Reactor system designed with the capabilities of a larger system but at a much lower cost and smaller footprint • F lexible, single-use vessel design is free of interfering inserts and probes • O perational control driven through on-board software interface • Bioreactor pump customization options allow high level of experimental control and flexibility • Ideal for stem cell research and d e velopment, cell and tissue culture , se e d development, educational applications and more • S u pports batch, fed batch and c o n t in u o u s b io p ro c e ss a p p lic a t io n s wit h a ddition of a cell separation d e vice Contact us to arrange a demo or trial at info@refinetech.com Also visit www. re finetech.com , or call (973) 952-0002, Ext 302. Drug Discovery Tutorial Figure 3. To get the most productive mouse chimeras, a combination of sperm chimerism and in vitro fertilization is necessary. Kenneth Albrecht, Ph.D. (kenneth. albrecht@taconic.com), is scientific program manager at Taconic Biosciences. Website: www.taconic.com.

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