Technical advances contribute to the study of genomic imprinting

Abstract

Genomic imprinting in mammals was discovered over 30 years ago through elegant embryological and genetic experiments in mice. Imprinted genes show a monoallelic and parent of origin-specific expression pattern; the development of techniques that can distinguish between expression from maternal and paternal chromosomes in mice, combined with high-throughput strategies, has allowed for identification of many more imprinted genes, most of which are conserved in humans. Undoubtedly, technical progress has greatly promoted progress in the field of genomic imprinting. Here, we summarize the techniques used to discover imprinted genes, identify new imprinted genes, define imprinting regulation mechanisms, and study imprinting functions.

Fig 1. Generation of normal mice using haESCs as gamete replacements.

(A) ICSI can generate normal mice. (B) The AG-haESCs support full-term embryonic development through ICAHCI, but with very low developmental efficiency. The generated mice are termed SC mice. (C) The PG-haESCs support full-term embryonic development through coinjection with sperm into enucleated oocytes, but with very low developmental efficiency. (D and E) The AG or PG-haESCs with deletions of two or three paternal DMRs efficiently generate normal mice through ICAHCI or ICPHCI. (F) The sperm-originated AG-haESCs with deletions in seven maternally imprinted regions (Nespas, Peg3, Snrpn, Kcnq1, Grb10, Igf2r, and Gnas) can be used to replace oocyte genomes for full-term development of bipaternal embryos using tetraploid complementation technology. (G) It is not clear whether AG-haESCs and PG-haESCs cultured in vitro can simultaneously substitute for paternal and maternal genomes and generate normal mice. The experiments that have been performed are outlined with a solid box; proposed experiments are outlined with a dashed box. Birth rate: percentage of transferred embryos. Surviving rate: percentage of born pups. AG, androgenetic; DMR, differentially DNA-methylated region; ESC, embryonic stem cell; Grb10, growth factor receptor bound protein 10; haESC, haploid embryonic stem cell; ICAHCI, intracytoplasmic AG-haESC injection; ICPHCI, intracytoplasmic PG-haESC injection; ICSI, intracytoplasmic sperm injection; Igf2r, insulin-like growth factor 2 receptor; Kcnq1, potassium voltage-gated channel subfamily Q member 1; KO, knockout; Nespas, neuroendocrine secretory protein antisense; Peg3, paternally expressed 3; PG, parthenogenetic; SC, semicloned; Snrpn, small nuclear ribonucleoprotein-associated protein N.

Fig 2. Combined applications of haESCs and CRISPR-Cas9 in functional studies of imprinted genes in vivo.

The sperm-originated AG-haESCs carrying deletions in both H19-DMR and IG-DMR combined with CRISPR-Cas9 editing technology can be used to study the function of paternally expressed genes and imprinting mechanisms and to trace the expression of imprinted genes. If AG-haESCs and PG-haESCs can be used to substitute for maternal and paternal genomes, respectively, and support normal embryonic development, it may be possible to simultaneously edit maternal and paternal alleles of the imprinted gene in the future (labeled by dashed box). AG, androgenetic; DKO, double knockout; DMR, differentially DNA-methylated region; H19, a long noncoding RNA; haESC, haploid embryonic stem cell; IG, intergenic germline-derived; PG, parthenogenetic, SC, semicloned.