Question

33. How can the function of an essential gene required for embryonic development be studied in an adult knockout mouse?

34.	How is the 5´ cap added to nascent RNAs?

31.	Describe some typical features of a restriction enzyme recognition sequence.

Answer 1

33. One way of studying the function of an essential gene that is required for embryonic development in adult knockout mice is by enabling sequence-specific suppression of virtually any gene using RNAi technique. In a study, Reversible suppression of an essential gene in adult mice using transgenic RNA interference, transgenic mice with TRE-driven Rpa3 shRNAs were used. The expression of TRE-driven Rpa3 shRNAs enforced a reversible cell cycle arrest. TRE or tetracycline response elements drive the expression of short hairpin RNAs (shRNAs). These aid in inducing reversible target gene suppression in mammals.

These shRpa3 transgenic mice showed rapid villus atrophy within 8-10 days of RNAi which was reversed as soon as the shRNA was withdrawn. This system provides a robust way of testing the function of essential genes in adult knockout models. (citation: https://doi.org/10.1073/pnas.1104097108)

34. The pre-mRNA is modified at both ends by the addition of chemical groups. The group at the beginning (5' end) is called a cap, while the group at the end (3' end) is called a tail. The cap and the tail protect the transcript. They also facilitate it getting exported from the nucleus and translated on the ribosomes (protein-making "machines") inside the cell.

The 5’ cap is added to the first nucleotide in the transcript during transcription. The cap is composed of a modified guanine (G) nucleotide. Also, it protects the transcript from being broken down. It also helps the ribosome attach to the mRNA and then translate it into a protein.

31.  Restriction enzyme recognition sequences have 1. a methylase sequence, and 2. restriction endonuclease. They are 4, 5 or 6 base pairs in length. Cleavage within the recognition sequence can produce
    1) blunt ends, 2) overhanging 3 prime ends, or 3) overhanging 5 prime ends.