Earticle

저밀도 리포단백질 수용체 관련 단백질 5(LRP5)는 간과 췌장을 포함하여 많은 조직에서 발현하며 아포리포단백질 E와 결합한다. 이와 같은 LRP5 유전자의 체내 기능을 규명하기 위하여 LRP5 유전자가 결손된 생쥐를 개발하였다. 먼지 LRP5 genomic DNA는 TT2 ES 세포로부터 분리하였으며 LRP5 유전자의 엑손 18에 neo 유전자를 삽입한 vector를 구축하고 TT2 ES 세포에 도입하였다. 178개의 G418 내성을 보인 세포 중 상동유전자 재조합에 의하여 targeting vector가 LRP5 유전자 위치에 삽입된 clone은 3개였다. 키메라 생쥐는 상실배기 수정을 ES 세포와 응집시켜 생산하였으며 생산된 키메라 생쥐는 C57BL/6 생쥐와 교미를 유도하여 heterozygous를 얻었다. 또한 이들 heterozygous간의 교배에 의하여 LRP5 유전자 결손 생쥐를 생산하였다. 이러한 생쥐는 LRP5 유전자의 체내 기능연구에 있어서 모델로 이용될 것으로 생각된다.

The low density lipoprotein receptor-related protein 5 (LRP5) highly expressed in many tissues, including hepatocytes and pancreatic beta cells, can bind to apolipoprotein E. To evaluate in vivo roles of LRP5, we generated LRP5-deficient mice. LRP5 genomic DNA was isolated from TT2 embryonic stem (ES) cells. Targeting vector was constructed to disrupt an exon 18 of the mouse LRP5 gene and transfected into ES cells. Three homologous recombinants at LRP5 locus were identified from 178 G418-resistant clones. Chimeric males generated by morula aggregation technique were mated to C57BL/6 female mice. After achieving germ-line transmission, LRP5+/- females were crossed with LRP5+/- males to obtain LRP5-deficient mice. One line of mice lacking LRP5 gene was confirmed by Southern blotting. Such knock-out mice may serve as an effective animal model to study in vivo function of LRP5 gene.

본 연구는 염증성 장 질환의 병리적 병변을 나타내는 IL10 유전형질 변형 실험동물 모델(IL10KO)을 이용하여 2주간의 운동적응에 대한 항염증 반응 및 대장수축력의 변화를 조사하였다. 그 결과 혈중 염증성 사이토카인의 변화에서 IL-6와 IL-1β 그리고 TNF-α에서 운동적용과 무관하게 정상군들에 비해 유전형질 변형 실험동물군에서 유의하게 증가하였으며, TNF-α의 경우 형질 변형군에서 운동에 의해 유의하게 감소하였다. 한편, 화학반응을 통한 장 절편 수축 력의 변화에서는 콜린성 반응과 질산매개성 반응에서 모두 운동여부와 무관하게 유전형질 변형 실험동물군에서 감소하였다.

This study examined changes in anti-inflammatory response and colon contractility for 2 weeks of motor adaptation using the IL10 genetically transgenic experimental animal model (IL10KO), which represents a pathological lesion of inflammatory bowel disease. As a result, in the change of inflammatory cytokines in the blood, IL-6, IL-1β, and TNF-α were significantly increased in the genetically modified experimental animal group compared to the normal group regardless of the exercise application. In the case of TNF-α, it was significantly decreased by exercise in the transgenic group. On the other hand, in the change of contraction force of the intestinal section through a chemical reaction, both the cholinergic reaction and the nitric acid-mediated reaction decreased in the genetically modified experimental animal group regardless of exercise.

The nul mutation of cardiac Na+-Ca2+ exchanger (NCX1) gene in mice caused death of embryo in utero at embryonic day (ED) 9.0-9.5 and this em-bryonic lethality appears resulted from abnormal heart development. In the present study, we in-vestigated whether transgenic re-expression of these lethal defects. Transgenic mice expresing the canine NCX1 in a cardiac specific maner were bred into the NCX1 knock-out background but did not prevent the fetal lethality asociated with the NCX1 nul alele. However, the NCX1 knock-out embryos with an NCX1 transgene sur-vived with heart beatings until ED 10.5 which was one day longer than the survival of the NCX1 knock-out embryos (ED 9.5). At ED 10.5, however, the partialy rescued NCX1 embryos might have succumbed to the lack of an organized vascula-ture in the yolk sacs. The placental labyrinth la-The transgenic re-expression of NCX1 rescued heart beatings and survived longer, but was stil insuficient for the mice to be completely rescued. Importantly, NCX1 was observed to expres in the yolk sac and the placenta of wild type mice. The results sugest that defects in extra-embryo-nic compartments are causal to the lethality, and that NCX1 may play an important role in es-tablishing vascularization in extra-embryonic tis-sues.

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.