Construction Of Pkm Knockout Mice Using CRISPR/Cas9 Technology To Study Tih Function And Mechanism Of Pkm Gene In Development

Glucose metabolism plays an important role in the development process,such as providing energy for various biological processes and participating in the regulation of epigenetic modifications.Early embryo before blastocyst mainly consumes pyruvate in the oviduct through oxidative phosphorylation process.When it develops to the blastocyst stage,it mainly utilizes intrauterine glucose to provide energy by glycolysis and oxidative phosphorylation while also increase oxygen consumption.Subsequently,the embryo is implanted into the wall of the uterus that lacks oxygen.The glycolysis process will dominate,and at this stage almost all pyruvate is converted to lactic acid.Finally,oxidative phosphorylation increases gradually in the stage of embryonic organogenesis and reaches a state of dynamic equilibrium with glycolysis.The changes of glucose metabolism types and the intermediates of glucose metabolism in early embryonic development are closely related to embryonic development.One of the key genes in glucose metabolism,pyruvate kinase(PK),is involved in the final step of the glycolysis process,catalyzing the reaction of phosphoenolpyruvate(PEP)to pyruvate.Genes encoding pyruvate kinase(PK)include pyruvate kinase M(Pkm)and pyruvate kinase LR(Pklr),each of which can produce two different isoforms.Two different isoforms of Pkm gene,Pkm1 and Pkm2,have different catalytic activities.To investigate the role of Pkm1 and Pkm2 in mammalian development,we used CRISPR-Cas9 gene editing technology to efficiently and rapidly construct Pkm1 and Pkm2 knockout mice.First,select the appropriate sgRNA sequences using Zhang Feng's sgRNA design website,and test them by detecting the cleavage efficiency on the embryo.Then,Cas9 mRNA and sgRNA were transcribed in vitro,and then two RNAs were injected into the zygote of C57/B6 by microinjection.Subsequently,the obtained zygotes were cultured in vitro to the 2-cell stage,transplanted into the oviduct of ICR surrogate mice,and the newborn mouse genotypes were detected,and Pkm1 and Pkm2 knockout mice were successfully obtained.We found that homozygous knockout of the Pkm1 or Pkm2 is fatal,but the time of death is different.Pkm1 knockout mice began to die after birth,the longest can only live to the sixth day,and the main death time is on the fifth and sixth days.In addition,when Pkm1 heterozygous mice were self-crossed,and the proportion of homozygous knockout mice was less than one-fourth in the offspring,indicating that embryonic death may occur.On the other hand,when Pkm2 heterozygous mice were self-crossed,no Pkm2 homozygous knockout mice could be obtained,indicating that the homozygous mouse died at embryonic stage.By separating embryos in different periods,we found that homozygous mice could not develop normally to 6.5dpc(days postcoitum).Due to the high expression of Pkm1 in heart and brain,a preliminary morphological diagnosis of mouse tissues was first performed by hematoxylin and eosin staining(HE staining).It was found that both the heart and brain tissues of Pkm1 knockout mice had obvious lesions.In addition,we found that adult Pkm2 heterozygous knockout mice had abnormal testis.At the same time,we detected the transcription level of metabolic related genes in the liver tissue of Pkm2 heterozygous knockout mice,and found that the expression levels of glycolysis-related genes such as glucose transporter(Glut1,Glut4),phosphofructokinase(Pfkm),hexokinase(Hk2)were significantly up-regulated;the expression levels of oxidative phosphorylation-related genes such as isocitrate dehydrogenase(Idh1)and citrate synthase(Cs)were significantly down-regulated.Pkm2 is mainly expressed in the mouse embryonic stem cell line(mESC),while the expression level of Pkm1 was very low,which was opposite of the results in adult muscle tissue.Since both Pkm1 and Pkm2 knockout embryos can develop into the blastocyst stage,we attempted to establish Pkm1 and Pkm2 knockout mouse mESC and found that Pkm2 knockout mESC could not be successfully established.However,we detected a decrease in the transcription levels of Pkm1 and Pkm2 in the Pkm2 heterozygous knockout mESC.This is consistent with the results in Pkm2 heterozygous mouse tissues,and the underlying mechanism needs further clarification.The Pkm gene involved in the glucose metabolism is closely related to mouse development.A preliminary study of Pkm1 and Pkm2 knockout mice revealed that the Pkm gene is critical for mouse development.But the function of Pkm1 and Pkm2 may different,the mechanism of Pkm1 and Pkm2 in early embryonic development needs further investigation.