Genome Editing in Mice Using TALE Nucleases.

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Creation of targeted genomic deletions using TALEN or CRISPR/Cas nuclease pairs in one-cell mouse embryos.

The use of TALEN and CRISPR/CAS nucleases is becoming increasingly popular as a means to edit single target sites in one-cell mouse embryos. Nevertheless, an area that has received less attention concerns the engineering of structural genome variants and the necessary religation of two distant double-strand breaks. Herein, we applied pairs of TALEN or sgRNAs and Cas9 to create deletions in the Rab38 gene. We found that the deletion of 3.2 or 9.3 kb, but not of 30 kb, occurs at a frequency of 6-37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to ∼10 kb can be readily achieved for modeling human disease alleles. This work represents an important step towards the establishment of new protocols that support the ligation of remote DSB ends to achieve even larger rearrangements.

Food-independent tendency to disadvantageous decisions in obese individuals with regular binge eating.

OBJECTIVE: The objective of this study was to explore whether or not obese patients with and without regular binge eating differ with regard to their decision-making abilities. METHODS: Decision-making was measured by using a computerized version of the Iowa Gambling task (IGT) in 34 obese patients with regular binge eating (BE(+)) and 34 obese individuals without binge eating (BE(-)) matched for age and sex. In addition, computerized versions of the Auditory Verbal Learning Test and the Corsi Block Tapping Test were administered. Participants further answered questionnaires concerning eating disorder symptoms (Eating Disorder Examination-Questionnaire) and depression (Patient Health Questionnaire depression scale). RESULTS: The BE(+) group reported more eating disorder and depressive symptoms than the BE(-) group but did not differ with regard to BMI, working memory deficits, depressive symptoms, somatic comorbidity (i.e., hypertension, diabetes, sleep apnea, hyperlipidemia, pain disorder), or education. Binge eating participants showed poorer decision-making abilities based on the total IGT net scores. However, they did not differ from those without regular binge eating in improving their choice behavior over the task. CONCLUSIONS: The group difference in total IGT net scores suggests more general, food-independent decision making problems in obese individuals with regular binge eating compared to those without. Treatment of obese patients with BED could be enhanced by training them to better control risky decisions, to delay gratification in an effortful way and to activate appropriate alternative behaviors.

Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA.

Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator-like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2-3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.

Performance in neurocognitive tasks in obese patients. Does somatic comorbidity matter?

The aim of the present study was to examine if obese individuals with obesity-related somatic comorbidity (i.e., hypertension, diabetes, sleep apnea, dyslipidemia, pain disorder) perform worse in neurocognitive tasks compared to obese individuals without any somatic disorder. Neurocognitive functioning was measured by a computerized test battery that consisted of the following tasks: Corsi Block Tapping Test, Auditory Word Learning Task, Trail Making Test-Part B, Stroop Test, Labyrinth Test, and a four-disk version of the Tower of Hanoi. The total sample consisted of 146 patients, the majority (N = 113) suffered from obesity grade 3, 26 individuals had obesity grade 2, and only 7 individuals obesity grade 1. Ninety-eight participants (67.1%) reported at least one somatic disorder (Soma(+)-group). Hypertension was present in 75 individuals (51.4%), type 2 diabetes in 34 participants (23.3%), 38 individuals had sleep apnea (26.0%), 16 suffered from dyslipidemia (11.0%), and 14 individuals reported having a chronic pain disorder (9.6%). Participants without a coexisting somatic disorder were younger [M Soma- = 33.7, SD = 9.8 vs. M Soma+ = 42.7, SD = 11.0, F(1, 144) = 23.01, p < 0.001] and more often female [89.6 and 62.2%, χ(2)(1) = 11.751, p = 0.001] but did not differ with respect to education, regular binge eating, or depressive symptoms from those in the Soma(+)-group. The Soma(-)-group performed better on cognitive tasks related to memory and mental flexibility. However, the group differences disappeared completely after controlling for age. The findings indicate that in some obese patients increasing age may not only be accompanied by an increase of obesity severity and by more obesity-related somatic disorders but also by poorer cognitive functioning.